Well, good early afternoon everybody.
What I’ll tell you today is a marathon that we’ve been running for the last almost 35 years:
The discovery and then the deeper exploration of the ubiquitin system.
And you heard a lot here on the central dogma of biology,
how the information that is embedded in DNA is then transcribed to RNA and then translated into proteins.
And there was very little interest along the years in how proteins are degraded.
People knew that proteins are degraded but learning from primitive systems
like the stomach where every protein that is going into the stomach is digested by proteins through amino acids.
People thought, well this may be also the case for our own body’s proteins.
And the idea was to discover the organ or the system that carries out this function.
But there was not really much interest,
especially not between the 60s and the 80s where much of the attention and focus was on the other side of the equation.
At that time I became a graduate student of Avram Hershko, a wonderful biochemist who became interested in this problem.
And because of some subtle points that attracted his attention, and then mine,
to what appeared to be a boring system but nevertheless evolved along the years into a very interesting platform.
So we are here in the very east side of the Mediterranean, in the beautiful city called Haifa that sits on the mountain,
on the Carmel mountain, and the Medical School is sitting right on the sea.
So if we look today 30 years later or 40 years later into what I call the destruction map of the cell,
it looks much more cumbersome than people ever imagined.
And I don’t have the time to go into all the details but there are 3 major systems that are all involved in destruction.
First is the apoptosis system that is involved in destruction of cells and we know how important it is, both for development
- for example just removing the interdigital tissue in order to generate the fingers but many other developmental processes –
and also in removing damaged, irreversibly damaged cells which protect us from numerous diseases
including malignant transformation.
Then we have the lysosome-vacuolar system that is involved not only in the degradation of extracellular proteins,
as was originally described by the late Christian de Duve, but also of intracellular protein - under stress mostly.
And a new field is now evolving in front of our eyes which is the field of autophagy
and there are different classes of autophagy and I will not go into it.
And this system, the autophagic vacuolar system, especially as related to intracellular protein degradation,
is involved mostly in bulk degradation of proteins, not in highly specific.
And then on the side sits here the ubiquitin system
that is involved in timed and programmed degradation of very unique and very specific proteins on which I'm going to allude.
All these systems talk extensively to one another.
Ambassadors or representatives or components of the ubiquitin system are sitting in the other systems,
as ubiquitin ligands, as some proteins, as another... and kinases and so on and so forth.
So there is a heavy talk/conversation/dialogue between the 3 systems that are well, well coordinated.
So as you can see today even the destruction that appeared 30 or 40 years ago to be kind of boring
and taken for granted now looks much more cumbersome.
So let's go to proteolysis.
The chemistry is very simple and it doesn’t matter where it occurs.
It's always in production of a molecule of water into a peptide bond in order to split it into 2.
And at the very end, once you split all the peptide bonds, we get back the amino acid that make the protein
and it doesn’t matter where it occurs.
It can occur extracorporeal in the gastrointestinal tract - it's outside the body, it’s a tube that crosses the body.
It can occur inside the body but outside of cells.
Like the blood coagulation system is a bona fide cascade of proteins,
it's an act one after the other and at the very end we are generating fibrin from fibrinogen.
And then it occurs inside the cells.
So you see they are kind of a ladder we are climbing, and as we climb the ladder specificity also is being attained.
So the commonality, the common base of all these systems, is this simple chemistry.
But they are very much different in the level of control.
But nevertheless, if we think about proteolysis - by definition proteolysis is destructive.
So always, always we need to protect ourselves from the destructive activity of proteolysis.
And it doesn’t matter where it occurs.
If it's in the gastrointestinal tract,
we don’t want the proteins that are secreted by the pancreas for example to digest the gastrointestinal tract.
So we have to protect it.
And we do it by secretion of a mucinous lining that protects the wall, the epithelium of the gastrointestinal tract,
from the digestive activity of the secreted enzymes.
And you just heard from Barry Marshall yesterday about what happens if there is an aberration in the process.
If there is a destruction in the continuity of the mucinous secretion, we are ending up with ulcers;
and you heard all the story of helicobacter pylori.
In the blood, for example let's take coagulation.
How we protect ourselves from untoward coagulation - by localisation.
So only when I'm wounded I'm secreting tissue factors.
And these tissue factors recruit blood coagulation factors
in order to make sure that the coagulation will occur only in the wound in order to prevent haemorrhage.
And not for example in other places that we don’t want it to occur,
like the coronary arteries where untoward coagulation does lead to one of the most common diseases in the western world,
which is myocardial infarction.
And in the cell - we shall see what happens in the cell, how the cell protects itself from the very activity,
or the digestive activity of the proteins.
Now the question comes, why at all do we need to degrade proteins?
Why not to synthesise them and to grow up and to attain our final weight at the age of 16 or 18
and live with all the reparative protein forever?
Why at all do we need to exchange them?
So we need to exchange them because of quality control, because proteins as you all know are very sensitive structures.
They are getting misfolded in nature, mutated, affected by post-translational changes like oxidation and so on and so forth.
So there is a constant need to remove damaged, harmful, inactivated proteins that are useless and to replace them with new ones.
We need to control processes:
so at that time, at that event we are removing healthy functional proteins that are not needed anymore.
Talk to Tim Hunt about cyclin - actually I think that Tim was one of the predictors of the profits of the ubiquitin system
by seeing this protein that cycles during cell cycle, that comes up and goes away at the proper time;
and it was just one single protein, while all other proteins were kept intact.
And then in order to maintain the differentiation and morphogenetic genetic state of tissues
we need to remove proteins permanently and in many cases - you heard yesterday the talk on teratocarcinomas, they are coming back.
So teratocarcinoma is a fine example of a tumour, of a malignant tumour where numerous tissues are coming back like teeth,
muscles, bones and so on.
So there is a dedifferentiation process.
So you see that there are many good reasons why we have to degrade proteins and we have to degrade them at quite a high rate.
Just to give you the number:
we are removing daily 6 to 8% of our proteins.
Some of them are removed several times an hour; they are cycling very quickly in order to attain control,
like transcription factor p53, MEK and others.
And some of them are long-lived like haemoglobin that lives a 120 days until it is removed by the spleen.
And in between you have ranges of different half-lifetimes,
but all in all I would say that within 3 to 4 months we are basically removing almost our entire protein.
Which is amazing, namely that the man that stands in front of you now on July 1st 2014 was not made of the same proteins
just nearly 4 months ago.
So it raises some nice philosophical questions:
who am I anyway if I'm not the same?
But it's going beyond this philosophical question.
Because the question that really should be asked is not only on the system that carries it out,
its control and biomedical implications, but also how is it that’s our software, which is our high functions of the brain,
is maintained - nevertheless we are exchanging the hardware.
And this is not a simple biological question.
But we shall not go into it, we shall just leave it, because there are some thoughts about what's going on.
So the founding father of the field we always give credit to the founding father.
Not only because we want to give them credit or respect them but because we are always on a continuum.
We never start anything and we never end anything.
We are taking something that was there, purify it further, distil it and donate it then to the next generation.
This is the way that science is going on.
I think that this is the reason why these people are so important to us.
Rudolf Schoenheimer, a Jewish scientist that lived here in Germany in Freiburg.
He was fired upon the rise of the Nazi to power, went to Columbia University to the Department of Chemistry of Hans Clarke,
where America at that time absorbed many of these Jews and even non-Jews.
And what Rudolf Schoenheimer did, he was using labelled amino acids, heavy isotope labelled amino acids and fed them to rats;
and he found that the amino acids are incorporated into proteins and then coming out.
So he found that the proteins are dynamic, which was really kind of a shock because everybody thought that proteins are static.
There were many Jews at that time - Albert Einstein among them, here in the Solvey meeting;
once he left Germany he never came back.
The term 'Hitler’s Gift' as you saw it on this book of Jean Medawar –
actually the widow of Professor Medawar who was himself a Nobel laureate for his achievement in immunology.
There were many reviews that were written on this Gift that Hitler gave to the west
in the form of this established wonderful scientists.
And among them Fritz Lipmann, the co-discoverer of I would say basic biochemistry;
he got the Nobel Prize for the coenzyme A, but numerous fundamental discoveries in biochemistry.
And then Rudolf Schoenheimer, Konrad Bloch, the father of the biosynthetic pathway of cholesterol, and many others.
And what Rudolf Schoenheimer did - this is an obituary that I found in the New York Times,
digging into the public library in New York - that his work helped establish
that the living body is a chemical laboratory where varied and complex transformation of matter are taking place incessantly.
So we are dynamic.
We are exchanging ourselves all the time and it's true not only for proteins but for lipids,
for small molecules and for other macromolecules, nucleic acids.
We are all moving all the time.
For proteins the field goes kind of dead, so he established the foundation,
the very fact that we are dynamic, but there was nothing known about the mechanism.
In the early ‘50s Mel Simpson at Yale found that protein degradation requires energy, which was a real surprise.
Here you see protein degradation, and he ... doesn’t matter.
The assay ones he trapped the oxygen from the incubator replaced it with nitrogen:
no ATP, no respiration, protein degradation stopped all together.
So he surmised that protein degradation requires energy but this didn’t make thermodynamic sense.
Because we are using proteins as a source of energy,
so why do we invest energy in order to degrade high-energy macromolecule into low energy like amino acid?
He guessed that it should be for control.
Think about driving a car downhill, there are 2 ways to do it:
either you turn off the switch and leave your leg off the brake; this is one way to drive but it will be a rather short ride.
Or if you want to survive it you shift the car to a low gear and put your leg on the brake generating heat and control the speed.
So you see that even if you don’t need to invest energy - well the analogy is not exact
but it gives you an idea that nature always pays with energy in order to control processes.
So this was the first milestone that picked up our attention.
And then came Christian de Duve, a wonderful scientist, just died in February 2 1/2 years ago.
And Christian discovered this organism, it is called the lysosome, he also shared the Nobel Prize for his discovery.
And the lysosome has proteases inside, protected by a membrane - again the idea of protection.
But then the question was how they bate, the proteins are getting into the teeth of the shark.
And there were several mechanisms; we know about them and they evolved along the years.
Some of them are receptor-mediated endocytosis of specific ligands like insulin, the PDGF molecule,
the cholesterol molecule that is being carried on the LDL particle - wonderful work by Mike Brown and Joe Goldstein from Texas.
Some are coming by pinocytosis, the cell is drinking the fluid around it,
and many other mechanisms that bring extracellular proteins into the lysosome.
How?
By including them in vesicles so vesicles are leaving the cell surface and migrating into the cell
and then they fuse with the lysosome and pour their contents into the lysosome.
It’s a well worked out studied mechanism - I will not go into it.
How about intracellular proteins?
Christian wanted to believe that the lysosome is the mother of all proteolytic processes.
And he said this is going by microautophagy.
So the lysosome pinches droplets of cytosol and converts them into vacuoles,
and these vacuoles then open up and pour their contents.
So there was this theory where ATPs were there.
We need to pump hydrogen ions into the lysosome; the pH in the lysosome is low by 2 units,
which is a hundredfold concentration of hydrogen.
Because the optimal activity... the proteins that are in the lysosome act only at low pH, they act optimal at low pH - why?
Again, in order to protect the cell, because if the lysosome explodes nothing will happen to the cell,
because the volume of the lysosome is very small and there will be neutralisation of the low pH
and the proteases will be inactive.
So think about evolution behind it.
So for years the field went into dormancy.
And because there was... people realised that proteins are degraded, they have a mechanism, energy is required;
everything went into silence which in many ways acted in our favour.
Because you are in a small country, limited resources, surrounded by big American sharks - so you want to be left alone.
But then people realised that this may not be the... that the lysosome may not be the organ - and why?
Because different proteins have different half-lifetimes.
This is just compilation of data.
And then Tim and Lee Hartwell and Paul Nurse came with the cyclin of the cell cycle and they said,
yes, proteins can be stable all along the cell cycle and all of a sudden they are degraded.
So you need specificity, the lysosomal mechanism couldn't explain specificity.
Because it's just bulk pinching off of droplets,
it's all the proteins that are in the site that are represented in this little droplet and are taken up and degraded.
So regulation and specificity looked to be out of the lysosome ability to handle them.
So people started to search.
We were not the first; there were several people, there were not too many.
One of them was Brian Poole, a wonderful scientist that worked along with Christian at the Rockefeller University,
and Brian did a simple experiment:
He inhibited the lysosome by using a base, ammonium chloride, which dissipates the intralysosomal pH.
And he showed within very simple experiments
- that I don’t have to the time to go into the details –
that chloroquine inhibits mostly the degradation of extracellular protein;
you can see here without chloroquine, and with chloroquine 68% degradation.
And while it doesn’t have... little affect say on the degradation of intracellular proteins,
so all of a sudden he said there must be 2 systems.
One, the lysosome that is inhibitable, and involved mostly in the degradation of extracellular proteins.
And then something else that is involved in intracellular proteins.
He didn’t shy out by poetically defining it in a beautiful way, saying the following:
“The exogenous proteins will be broken down in the lysosomes,
while the endogenous proteins will be broken down wherever it is that endogenous proteins are broken down.”
Somewhere, go and find it.
And he did go to find it.
Unfortunately he died of complications of metabolic disease I came to know him during my graduate studies.
He was a very generous competitor I must admit, made all his tools and knowledge available to us.
But that’s what happens.
And along with Avram we launched our journey, we started with a reticulocyte which is a terminally differentiating red blood cell.
It's another lack because for years people didn’t believe us;
and if they did believe us they said this is not biology - this is the corner of biology, it’s a terminally differentiating cell.
So that again gave us some time to dig into the system.
The reason we decided to go to the reticulocyte is because the reticulocyte doesn’t have lysosomes,
it ejects them out during its maturation.
And then it continues to degrade further its other machineries to live behind very primitive cells
- basically a vacuole full with haemoglobin, with glycolytic enzymes, in order to generate ATP,
gluthathione in order to keep the reduced state of many other proteins –
but very primitive cells.
So this is a kind of a rapid differentiation.
So nature provided us:
there was no siRNA, we couldn't silence any proteins at that time.
So we went and we published the first paper in ’78 in a very awkward journal.
Yesterday Randy Schekman commented on the new culture, I will not go into it,
but this is a journal that nowadays will take you nowhere.
It took us to Stockholm.
It was cited by the Committee which tells you very briefly
that it doesn’t matter WHERE you publish it matters WHAT you publish.
But let’s forget about this one.
The paper was important in the sense that immediately it left us without a paradigm.
So already at the very beginning we were left without a paradigm.
And the reason was that indeed in the reticulocyte high speed supernatant we found ATP-dependent proteolytic activity
and then like good biochemists we started to fractionate it and it was fractionated.
And instead of the classical tango
- that proteins are dancing which is a tango of 2 where you have a protease and a substrate –
already in the first step we fractured it into two.
So fraction one doesn't matter, it depends on the column that you use.
Didn’t have any activity we just split it.
Fraction 2 didn’t have any activity or little and we had to combine them.
So this is a tango of 3 already:
2 fractions that are needed to degrade the third partner.
Now remember these 2 fractions contain each 10,000 proteins, because it's the cell lysate that was.
So then we started to further fractionate because nobody told us that fraction 1 has only one component.
And the same for fraction 2.
5 years of work.
When I left the lab to go to MIT to a postdoc I left behind about 14 different factors
that are all needed in order to degrade one single protein, but most importantly we knew what they were doing.
So this was the more difficult part.
It was not the fractionation, but assigning each a role and put them in a row to know who is first.
So we started to write the alphabet of the system.
Because of a lack of time I’ll show you the alphabet of the system but in a summary and then I will jump quickly to medicine.
So the system is now known to be composed of close to 2,000 proteins - it's almost 10% of the human genome.
And I’ll just show you the principle components:
Here you have a substrate, this is the victim to be degraded.
Something typically has to happen to the victim, it has to be oxidised, phosphorylated, hydroxiprolinated
- something needs to happen to it in order for a policeman to identify it.
The policeman is called ubiquitin ligase.
It’s a huge family that’s made of sub-families
- there are almost 1,000 ubiquitin ligases –
so half of the family of the ubiquitin, the big ubiquitin family, is made of recognising the enzymes.
This is a critical element of the system:
It recognises the substrate to be degraded and it binds it.
That’s what it does.
Meanwhile a small molecule called ubiquitin is being activated
by a single component in the database called E1 or ubiquitin activating enzyme to high energy.
We jump it to high energy - I don’t have the time to show you all the biochemical details, but it's beautiful biochemistry.
And from there it jumps to a second enzyme, it’s a transmitting, it’s a conveyor called E2.
There are about 50 E2 in the database.
And from there it goes to the substrate, to mark the substrate.
So now we have a substrate with something happening to it and it's marked, but it's still intact.
Only now, following the ubiquitination
- actually it's multiple ubiquitination, so it's not a single molecule of ubiquitin –
the first bind to the substrate, the second to the third, the third to the second, and so on and so forth.
Then come the scissors.
And the scissors will recognise only ubiquitinated proteins, they will not recognise naked proteins.
There is one exception which is ornithine decarboxylase;
I don’t want to talk about it, but this is basically the only well-established known exception.
All the other proteins have to be ubiquitinated.
And the protein recognises this ubiquitin, it doesn’t recognise the substrate;
it brings the substrate, however, close to the protease.
The substrate is opened up by the activity of ATPase; it’s a huge substrate,
the structure of which was deciphered by Robert Huber and Wolfgang Baumeister and others
in the Max Planck Institute in Martinsried near Munich, it’s a beautiful structure.
And it's opened up by the activity of ATPases that are here in the upper and lower complex.
It is composed of 2 sub-complexes, the 19th and the 20th, it's a huge, it’s a 2-Megadalton, 2 million-dalton protease.
The active chamber is here, so the proteolytic activity occurs here in the 20th complex, in the alpha rings.
You see there are 4 rings, 2 alpha rings at the periphery and 2 beta rings in the middle, and there the activity occurs.
And there is a degradation of the substrate to peptides, not to amino acids,
and these peptides are also part of the immune system serving as MHC class I molecule peptides
- but again I don’t want to talk about it.
The system got complicated along the years because each ubiquitin is bound to another,
typically via one lysine which is lysine 48 but there are others, 7 lysines that can also bind ubiquitin.
And gradually a whole language evolved around the system.
And again I'm sorry that I don’t have the time and the language is complicated
because the ubiquitin can be bound via a single lysine or there can be 1 ubiquitin that 2 ubiquitins will bind to it
- so it's all 3, it can be like a chandelier.
And then there are linear chains, that each ubiquitin is attached to the other:
head to tail, head to tail.
And there are monoubiquitinations and oligoubiquitinations.
And then the whole family of proteins, of cousins of ubiquitin, which are called ubiquitin-like proteins, were discovered,
SUMO, Rub, NEDD and so on; and the whole family increased dramatically.
Not only in the number of the members of the family but also in the way they link to one another.
And it turned out that what we discovered is a proteolytic machinery,
but what we discovered more than that is a novel form of post-translational modification
that serves numerous functions, like phosphorylation.
But the beauty of the system is that we didn’t want to discover the ubiquitin system,
we didn’t have any intention to discover, we asked the biological question.
So we came from the very bottom and gradually we peeled it off like an onion.
And then the informaticists came and other people came and added up on top of it.
So I think this is a more elegant and beautiful way to approach biology asking a question rather than screaming;
but obviously both approaches are legitimate and people can come to wonderful conclusions from both directions.
But we came from the bottom, from the biology.
The homology tool phosphorylation - Eddy Fisher is here really striking.
You see the ligases, the 1,000 ligases that modify ubiquitin, the 600 kinases.
Ubiquitin can be deubiquitinated, can be recycled.
Phosphatases will take phosphate off.
Phosphates generate a signal, an output signal to be recognised by downstream element
- the wonderful work of Tony Pawson and Tony Hunter.
And ubiquitin also, as I said in one case it’s a signal to the proteasome
but depending on the structure of the chain it’s a signal for numerous downstream effectors.
And, most importantly, the 2 systems talk to one another.
So some proteins, as I showed you in the scheme, will be degraded only following phosphorylation.
Let me jump very quickly in the last 2 minutes to medicine.
With 2,000 components cannot be that aberrations in some of them will not lead to diseases.
And I’ll show you one example but the principle is the following.
Proteins are degraded, 2 things can happen:
either one degraded excessively or one is not degraded;
they either go below their steady state or accumulate above their steady state, and here you have a disease.
So there are many diseases.
Basically all the neurodegenerative diseases have the ubiquitin system involved,
either as a primary culprit or as a secondary culprit.
And if you look at brains of Alzheimer’s patients, this is early-onset Alzheimer's patients,
you will see accumulation of constipated conjugates.
So these conjugates are ubiquitinated but the proteasome is constipated, it cannot degrade.
But you will see these ubiquitinated proteins in numerous diseases.
But let’s go not to quality control but to control of processes; and cancer immediately comes up.
Why cancer?
Because either excessive degradation of the tumour suppressor like P53 that occurs in human cervical uterine carcinoma
- the mechanism of which was beautifully unravelled by Harald zur Hausen –
will lead to cancer, because we are removing from the cell the tumour suppressor protein.
How does it occur?
The virus enclosed in an early gene called E6 heterodyne rises to P53, the ubiquitin system looks at it,
it finds something foreign and it trashes it.
What was the purpose of the virus?
Exactly that:
to mislead the ubiquitin system to remove a tumour suppressor - but we shall not talk about the evolution of viruses.
On the other side the very same.
An oncogenic protein that will be under degraded or a growth promoting protein
that will be under-degraded will also lead to malignant transformation.
Think about the EGF receptor that is mutated in numerous breast cancers.
It signals to the cells nonstop.
It cannot bind the ligand anymore, because the engagement of the ligand will lead to its internalisation and degradation.
And we have some solution for it:
the antibody Herceptin, that will cluster it on the cell surface, but this is not an ultimate solution.
So let's drug it now; we have disease, we can drug it and with that I'll end up.
The first drug
- I’ll show you just one drug but there will be many future ones because time is coming to an end –
very successful drug, it's an inhibitor of the proteasome.
People thought that this was unrealistic to inhibit the proteasome because the whole ubiquitin system will be clogged behind.
Then why I'm coming and telling you all these stories here, how important or not important it is?
But the partial inhibition of the proteasome does lead to a very beneficial effect in one disease
which is called multiple myeloma.
This is the inhibitor, Bortezomib, it’s a tripeptide and it's a boronic acid derivative.
And here you have a patient with multiple myeloma - multiple myeloma is a malignant disease.
It’s a B-cell leukaemia where there is a monoclonal expansion of a B-cell in the bone marrow.
The patients are suffering from suppression of all other elements of the bone marrow:
red blood cells, anaemia, white blood cells, infection, platelets, haemorrhages, fractures
because the mass of the tumour cells is pressing.
And here you can see a nice treatment:
The disease gallops, the B-cells secrete immuno-globin, chemotherapy started, partial effect, relapse, ... (inaudible 31.11).
And here you can see the bone marrow of the same patient.
Heavily malignantly floated bone marrow with 41% cells of malignant cells.
Following treatment less than 1%, then rejuvenation of the bone marrow with its normal progenitor.
And the very last one, you can see an X-ray of another disease, which is a cousin disease, non-Hodgkin lymphoma.
You can see the tumour sitting here at the gate of the right lung, and significant regression following treatment.
You can see here the ribs, the sternum, the vertebra, the shoulder and so on.
So you can see we had nothing to do with these drugs, no rights even, no royalties, nothing.
But how basic curiosity in just unsolved problems leads at the very end not only to the discovery of an important system,
but also to huge benefits to human kind.
Thank you very much.