I have the pleasure to introduce the next speaker, Professor Aaron Ciechanover from the Technion in Haifa, Israel.
He received the Nobel Prize in chemistry together with Avram Hershko, who talked yesterday,
and Irwin Rose for the discovery of ubiquitin-mediated protein degradation.
Professor Ciechanover has been in Lindau every year since he got the prize and he obviously likes Lindau.
And we want to thank him for his sympathy for the meeting and the support of the meeting.
His lecture today is on the dynamics of our proteins from basic mechanisms to the patient bed.
Aaron.
(Applause).
I would join Richard Roberts' recommendation as for future projects or research of students.
And by myself I would think, if I had to choose today, I would have walked into microbiology and virology,
not only because it's fascinating, but because it's so awfully important.
And if we think of just the past 30 years of legionella disease and ebola and avian flu and AIDS,
we realise that we are living on a very thin margin.
And some mutations in all those organisms would have caused them to be much more virulent
and may have brought us back to middle ages and to black plague.
And somehow we were under the illusion that we defeated infectious diseases.
And, as Richard mentioned, governments stopped funding it and it was just an illusion.
So let me start with my own talk, and I try not to overlap with my past mentor's talk yesterday,
and I'll touch on the periphery of what he talked to you yesterday.
I'll go a little bit to the history of the field of proteolysis in the ubiquitin system,
not just to pay credit to the people that established the field, because I think that we play just,
not a marginal role but we play the very defined role in the middle.
But also to show you that once you are walking into the lab and you are up to a new project,
you should really know what other people in the field have done.
And you will see that always there are many milestones around
and all that you have to do is just to combine threads and ropes and then, based on it, build new knowledge.
And then I'll jump over the detailed history of the discovery itself and go directly to its application into medicine.
And I'll show you how drugs are being developed, current drugs and also future drugs.
Where the pharma companies are going with it.
So we are all about proteolysis and proteolysis is an old process, we know it very well, that we have to hydrolyse amino acids.
And it occurs in the body in different organelles and it's all about topology in the gastrointestinal track.
It's a simple process, every protein that goes in is degraded non specifically.
We remove antigenicity, we derive energy from nutrients and we know all about it.
Once we are moving into that body, we are still in the circulation, extracellular, intracorporeal,
they are very limited proteolytic system that are highly specific, one was mentioned yesterday by professor Robert Huber,
the blood coagulation system, is a bona fide cascade of proteolytic events.
But at the end fibrinogen is converted to fibrin and we are generating the blood clot,
we know what happens in pathologies, overcoagulation, myocardial infarction, undercoagulation, a whole set of genetic diseases, haemophilias,
but these are limited processes.
Now we move into the cell and in the cell there are several proteolytic processes and when I was a graduate student,
I joined Avram lab and we were interested in this process and it turns out that the process is extremely extensive.
So what happens in our body is we are turning over our proteins,
we are degrading them and resynthezise them in an amazing pace and the pace is,
about 5 to 7% of our proteins are degraded daily and resynthetised, renewed, and the question is why it happens.
And there are several reasons why it happens, one reason is obviously quality control.
Many of our proteins get misfolded in nature because of many reasons.
For example the high temperature that we are living, 37° is a deleterious temperature for proteins, high oxygen, 21%.
Obviously there are many good evolutionary reasons why we are living at 37° and why we are living in an atmosphere of 21%.
But on the other hand, this is deleterious to proteins and we have to exchange them.
We control processes, you heard the lecture, the wonderful talk of Tim Hunt, Avram's yesterday.
There are very good proteins, but we don't need the process anymore.
Cell division, the cell, the entire cell cycle is controlled by a whole series of brakes and accelerators
that have to be removed at certain time points, and that's another reason why to degrade proteins at a certain time point.
And obviously, in order to keep the differentiated state and morphogenetic state of tissues,
we need to remove certain proteins permanently at certain times, a process that is being reversed in malignancies.
Now, let's go a little bit to the history.
And in my opinion the founding father is Rudolf Schönheimer,
a German Jew that went to the United States upon the rise of the Nazis to power,
and he worked at the Rockefeller University in New York.
And he, along with Harold Urey, used a very simple method, he used labelled amino acid,
fed it into rats and found that the amino acid goes into proteins and then comes out.
So he concluded that proteins or body constituents are in a dynamic state
and he published a small monograph called on the dynamic state of body constituent.
And this was published in 1942 at Harvard University press.
And Rudolf Schönheimer died untimely, this is the obituary taken from the New York Times in '42
and it's clear that that's what he did
and it says that his work helped establish that the living body is a chemical laboratory
where varied and complex transformations of matter are taking place incessantly.
Then, another milestone that was neglected along the years was of Mel Simpson at Yale.
And Mel Simpson found something that was thermodynamically paradoxical, that protein degradation requires energy.
And, as Avram explained to you yesterday, it didn't make any sense because proteins are high energy ingredients,
so why to invest further energy in order to degrade them to amino acids,
but nevertheless, this is protein degradation in Mel's experimental system.
And when he changed the atmosphere in the incubator from oxygen to nitrogen, protein degradation ceased altogether.
Obviously he didn't understand the explanation, he didn't have the ubiquitin system at hand and he didn't know what's going on.
But he concluded that this is an energy requiring process.
Then came a wonderful scientist, Christian de Duve, and he discovered the lysosome
and he shared the Nobel prize in the mid '60's, along with others that discovered other organelles of the cell.
And this is the lysosome.
And the lysosome is an organelle secluded by a membrane and contains inside its lumen proteases.
And this provided the explanation that the mechanistic base for protein degradation.
So people knew that proteins are degraded and here came the mechanism.
And the field actually between the mid '50's, when this discovery was made, to the mid '70's fell into dormancy,
because people thought, well, there is nothing to study anymore.
And the problem was only how proteins get into the lysosome.
So for extracellular proteins like glycan, like the LDL core particle or the insulin, they are binding to receptors,
going via endocytosis, receptor mediated endocytosis, and then are transported by a series of vesicles into the lysosome,
these vesicles fuse, they pour their contents and their proteins are degraded.
For intracellular proteins, microautophagy, you see this pinching of small droplets of the cyctosol, and these contents fuse,
they make vesicles and then they fuse, pour their contents and degraded.
So both extracellular proteins and intracellular proteins were assumed to be degraded by the lysosome.
And then doubts started to be casted, this taken from a review of Fred Goldberg,
Bob Shimky actually preceded him by this assumption.
Here you can see the different proteins, doesn't matter even which, have different halflife times,
like some proteins are living in the cell for merely few minutes, like ornithine decarboxylase.
Other are living for more than 15 hours.
And you heard that even for a single protein, like cycline, you heard it from Tim Hunt,
for certain part of the cell cycle they are very stable, and then, all of a sudden, they are gone.
This couldn't be explained based on the lysosomal theory,
because the lysosome by pinching of droplets of the cytosol should degrade all the proteins at the same very rate,
because proteins are not resistant to the activity of the lysosomal proteases.
So these findings of the different various halflife times, and even changing halflife for specific proteins,
could not be explained based on the lysosomal theory.
And I would say that one of the last nails in the coffin of the lysosome is the main organelle
that targets intracellular proteins came from Brian Poole.
And Brian Poole was working actually in the laboratory of Christian de Duve at the Rockefeller University,
so it's very nice how things cohere.
And he did a very elegant experiment that I will not tell you all the details of,
but he was able to inhibit lysosomal degradation by using a simple drug that we are all using against malaria,
it's called Chloroquine, it kills the malarial parasite.
And Chloroquine is known to increase the intralysosomal pH,
I fail to tell you that the lysosomal proteases act optimally only at a low pH,
which is provided by the acidic environment in the intralysosomal milieu.
It's about 5 - 5.5 and while you are treating cells with Chloroquine, Chloroquine is the base,
it goes in to the lysosome, gets protonated and dissipates the low intralysosomal pH, so the lysosome is shut down.
And what Brian Poole did, he asked himself whether, when he shuts down the lysosome,
both extracellular and intracellular proteolysis are being inhibited.
And the answer was no, only extracellular proteolysis is being inhibited
while there is no effect on inhibition of the lysosomes on intracellular proteolysis.
And he summed it up in a beautiful poetic way that basically for us, though I cannot link things datewise,
was kind of a green light: Yes, you can go ahead and search what you are looking for.
And the summary goes as follows: Exogenous proteins, meaning extracellular proteins will be broken down in the lysosome,
because the degradation is inhibitable by lysosomotropic agents.
While endogenous, intracellular proteins will be broken down wherever it is that endogenous proteins are broken down,
wherever it is.
So we were after the 'wherever it is'.
And I was a graduate student with Avram, and Avram told you yesterday the history,
the fractionation of the reticulocyte lysate, beautiful elegant biochemistry at its best.
And we found a conjugation machinery that takes protein for the final terminal destination.
I just show you the mechanism in a snap shot, I will not go through the details.
It's basically a 3-step mechanism and I tell you also why it was a very important one.
The first, the target protein is being recognised by an enzyme called ubiquitin ligase, this is the enzyme that ligates ubiquitin, the death tag to the protein, there are about 1,000 ligases in the data base.
Just to be accurate, there are about 22,000 and few hundred proteins in the human genome,
not counting obviously gene splicing products like MHC class I molecules, T-cell receptors, antibodies, but straight genes.
So the ubiquitin, just the ligases comprise about 5% of the total human genome.
This is a specific recognition, only specific substance has to be targeted or recognised by specific ligases.
In parallel, so this is the first step, recognition.
Second step is tagging, here is ubiquitin, it's being activated by an enzyme called E1, 1E1 in the human data base.
Activation, that step requires ATP, this solves the problem why we need ATP, I will not go through the details.
And then, via E2, so it's a cascade reaction, it's transfer of ubiquitin from one enzyme, from E1 to E2,
and then to the substrate we are building polyubiquitin chain.
And this polyubiquitin chain, so the second step is tagging, recognition, tagging and now degradation.
Here comes the protease, it's a huge 2 megadalton protease that recognises only polyubiquitin proteins
and send them to the final destination.
The polyubiquitin chain binds, basically the polyubiquitin chain is a recognition element in trans,
it binds the substrate to the protease, the substrate get unfolded, injected into the proteolytic core
that you heard yesterday, partially by professor Robert Huber and degraded into peptides.
The beauty of this mechanism and why it was away from any paradigm, that it allowed the process to occur in one compartment,
because otherwise proteases and substrates cannot live in the same compartment because it will be chaotic,
if not destructive to the cell.
So we have kind of a barrier here, the protease will never recognise the substrate unless the substrate is being tagged.
And this immediately shifted all the attention in the field to why certain substrate are being tagged at a certain time point.
And the proteolytic process remained kind of in the backyard.
So the specificity and the recognition became immediately the focus of research in the field,
but it immediately explained why everything can occur in the same compartment.
While in the lysosome we needed the fence, the membrane to separate between the shark and the bait.
So this is the ubiquitin system.
Let's go now, and the ubiquitin system, as I told you, is hierarchical, single E1, transfers ubiquitin to about 50 E2s,
each E2 talks to about more E3's, there are about 1,000 in the data base
and each E3 will polyubiquitinate a certain subset of substrate
and that once polyubiquitinated will go to the proteasome to be shredded.
Now, let's go to diseases, let's jump to the other part of the talk very quickly.
And normally, this is the ubiquitin system, proteins are in a steady state and we are facing a problem in one of two cases.
Either a protein is overly degraded, excessively degraded or it's underdegraded.
Let's say that the ubiquitin ligase is overexpressed,
it will take the protein down or there is a mutation in ubiquitin ligase or in the recognition motif of the substrate,
the protein will accumulate and in both cases we are into a problem.
Now, we can go back to the old slide that I showed you and can tell you
that in each section of the functions of the ubiquitin system there was a whole group of diseases discovered.
And I'll go briefly through just two of those functions of the ubiquitin system again,
the quality control and control of processes.
Quality control will remove the nature misfolded proteins.
Basically in all neurodegenerative diseases you will find accumulation of proteins
that should have been degraded and are not degraded.
And you will find it in Parkinson, you will find it in polyQ expansion diseases, in Huntington disease,
spinocerebellar ataxia, Kennedy disease and so on and so forth.
This is just one case.
We learned a lot about it, not from the family onset diseases but rather from the genetic diseases, that are more rare,
in this case you have Alzheimer.
These are sections of Alzheimer brains from patients, it was done in Holland by Doctor Van Leuven, from Amsterdam.
And these are, you see here staining for ubiquitinated proteins
and in this case the patients have a transcript misreading of ubiquitin.
So ubiquitin is being translated with an extension of 20 amino acids, it's a misfunctional ubiquitin,
it can be conjugated, it cannot be conjugated, the cell sense less of ubiquitin and a disease ensues.
Avram mentioned yesterday, I think in the discussion or during his talk, Parkinson,
there is a juvenile form of Parkinson that erupts at an early age, in the third and fourth decade in life.
There is a protein called Parkin, Parkin is a ubiquitin ligase, Parkin is mutated,
probably a certain protein that should be targeted by Parkin is not targeted, it's accumulated and it becomes neuro toxic.
So here you can see how the defect in the ubiquitin system leads to development of neuro degenerating diseases.
Let's go to control of processes and the best studied diseases or set of diseases are malignancies.
And here I shall go into more details and show you really how drugs, how people are thinking about drugging the system.
Now, let's go to cancer.
And we can, again Avram showed it to you yesterday, we can imagine cancer as a balance of kind of quality control in the cell.
I mean, all the time we are mutating our cells, we are causing them damage,
we are stressing them and they are balanced mechanisms that have to take care of it.
The most studied one is obviously p53 and I'll come back to p53.
53 is the genome guardian, it's kind of checking all kinds of stress and damages
and it will initially immediately stop the cell cycle, try to repair the damage, and if the damage cannot be repaired,
it will induce apoptosis.
So it's kind of a timelined reaction.
And we can imagine that if there will be an increase degradation, overdegradation of a tumour suppressor like p53,
the cell will be immediately exposed to malignant transformation.
And that's what exactly occurs in human papilloma virus induced uterine cervical carcinoma,
the virus encodes an oncogene called E6.
This oncogene heterodimerises to p53, the ubiquitin system is misled to think,
to identify this heterodimerise p53 as a denatured, wrong foreign protein,
disposes it and the cell is out of p53 and out of the p53 checkpoint pathway.
And the viruses now can go, this is an early gene, E6, and now the virus can go on and integrate its DNA into the cellular DNA.
If the virus wouldn't have gotten rid of p53 and would have gone directly for the integration event,
the p53 would have jumped up because it would have sensed some DNA damage and would have tried either to repair the damage or,
if failing, to apoptose the cell.
So the virus has evolved, think about it evolutionary,
a strategy to remove the alarm system from the cell in order for it to allow it to propagate and to replicate,
very elegant mechanism.
On the other hand, oncogenical growth promoting protein that will not be degraded, like a mutated EGF receptor,
that will keep on signalling to the cell non-stop, or beta-catenin in the wind signalling pathway,
that will not be degraded, will keep on signalling to the cell non-stop, will cause cancer.
We know that the machinery that degrades beta-catenin is defective in many, many cases of colorectal carcinoma,
malignant melanomas and many others, breast carcinomas with EGF receptor and so on and so forth.
So we know that under-degradation of a growth promoting protein, over-degradation of a tumour suppressor, is bad for us.
Now, if we are thinking of drugging the system, mutations are more difficult to treat.
I mean, loss of function is always in concept more difficult to treat than gain of function.
Think about pharmacology today, we are treating hypertension by beta blockers.
We are treating all kinds of diseases by inhibiting processes that are working in excess than interfering with mutations.
You know, treating mutations typically throws us into the big swamp of genetic treatment
and here the ubiquitin system is obviously not an exception.
But let's go to drugging of the system and we shall go step by step.
And the first inhibitor that was developed was an inhibitor to the proteasome.
And this didn't make any sense.
I mean, drugwise it didn't make any sense because if the ubiquitin system is so important
and if we are going to inhibit the proteasome, then the patient will, if not dying, at least develop severe side effects.
But it did work and it did work magically and because some cells are more sensitive even to partial inhibition than others.
And I'll show you, this is the drug, Avram mentioned it yesterday,
it's Velcade and it's a specific inhibitor of the active side of the proteasome.
And it's an aldehyde boronic acid derivative and it's being used now in two diseases but it's a new drug.
And it was just approved by the FDA a few years ago and it's being expanded all the time.
Here is a patient with multiple myeloma, for those of you who are not haematologists or in the field,
it's a monoclonal expansion of an immune cell in the bone marrow.
And the immune cell suppresses all the normal progenitors in the bone marrow, it's a deadly disease.
All the progenitors are not there, because of the pressure there are vertebra fractures, long bone fractures,
it's a deadly disease, very bad disease.
And here you can see this, in this case the oncologists are lucky because they can follow a biomarker
which is not the case in many malignancies.
Here you can see the galloping immunoglobulin that is secreted by the cell, conventional treatment started,
the patient responds partially, relapses and here started treatment with this inhibitor and the patient responds favourably.
About 5% of the patients go into a very long remission which was unheard before.
35% respond favourably, 60% don't respond at all.
And the challenge is now, is to genetically profile these patients and to understand the differences between the responders,
the partial responders and the non-responders.
But this belongs to what we call now, in modern medicine, personalised medicine.
We are moving from general medicine into personalised medicine
and into a completely different way of how we look at patients and the response to treatment.
This is the bone marrow of the patient, you can see without being histo- or hematopathologist,
you can immediately tell that this bone marrow is malignant, inhomogenous, full with malignant cells.
And the following treatment, you can see that the malignant cells went down from 40% to 1%.
And the bone marrow is enriched again with a normal progenitor.
This is another patient with Non-Hodgkin lymphoma, it's another disease of the immune system and without going into details,
you can see here the tumour that sits in the right hilum of the lung.
These are the big vessels, the sternum, the vertebra, the spinal canal, the scapula, the muscle of the shoulder, the ribs.
And you can see here the tumour that regressed following a treatment.
Now, let's go to the next, to the future.
And what I want to do now, in the last 5 minutes of my talk,
is just take you very quickly to the future and show you how we think.
So the first treatment was clearly non-specific,
it produced apoptosis on the cell but it doesn't make sense to drug and to inhibit the proteasome and many diseases will not.
So people are trying to go more and more into specific treatments and I'll take you through the specific treatments.
So we know that in the cell one of the quality control mechanisms are chaperons.
And chaperons are proteins that are trying to refold misfolded proteins and if they are unable to do it,
they deliver them to the ubiquitin system for degradation.
But from time to time they play the wrong role.
Instead of, on one hand, they cannot refold the proteins that they should refold,
but on the other hand they bind them irreversibly and cannot deliver them for degradation.
So we get stuck and the mutated proteins get protected from degradation.
So basically we can say that these HSP proteins, these chaperones, they are acting as evolutionary capacitors of mutations,
they just augment mutations.
So the idea will be to develop dissociators that will take the mutated proteins away from its protector.
So that's what people did and there are several drugs already in trials, and one of them is Geldanamycin,
it's too toxic but the concept is there.
Another one is 17-AAG and I'll show you just one example.
And here you can see the HER neu receptor, the same one that is targeted by Herceptin,
is here fluorescently labelled and following treatment with the dissociator from the chaperone,
the fluorescent is gone, the receptor has been degraded.
And you can see here actually the real biochemistry of how it works, it's really beautiful.
This is the receptor, the protein, this is the activity of the receptor, the tyrosine protein kinase.
So here we are measuring with specific antibodies that recognise phosphor tyrosine.
And you can see that once we are increasing the concentration of the dissociator, this guy here on the left,
you are increasing the amount of ubiquitin conjugate, so you send the receptor to its final destination.
The protein goes down, but what's really impressive is that the activity is going down.
The phosphor tyrosine kinase activity is almost gone.
Now let's go to patients, I mean we are moving to patients, these are a little bit not so nice diseases but we are in medicine.
So this is a patient with spread squamous cell carcinoma of the face, you see 40 days later the face is much better,
this is just an ulcer, so the tumour was ulcerated, was necrotised.
And 80 days later you can all tell much improvement, with single modality therapy.
So here you have a mechanism-based treatment,
we release a mutated active protein from its capacitator and send it for destruction and the patient improve.
Let's go to the next generation of drugs,
which is really the future and the human experiments are still at the very initial phase.
Let me go back to p53, as I told you p53 is a critically important protein.
In 50% of all tumours across the board it's mutated and in another 50% of the remaining 50%, namely 25%,
it's underrepresented, it's overly degraded.
So it's a huge major player in malignant transformation.
And every single stress, ribonucleotide depletion, DNA damage, nitric oxide, will cause p53 to jump up.
If it's not mutated, I'm talking only on the wild type, it will immediately stop the cell cycle,
because a cell that is stressed cannot proceed.
Will induce DNA repair, and if not, will induce apoptosis.
And let's forget about senescence.
So p53 is our good guy, oncogenic stress increases, activates p53 to prevent cancer, it's oversimplified but it's awfully true.
Now, in those cases the p53 is the wild type but it's nevertheless underrepresented,
it's underrepresented because its ubiquitin ligase, its executioner is over expressed.
Basically the Hdm2, which is the ubiquitin ligase, the executioner of p53, becomes an oncogene,
it's up-regulated in an uncontrolled manner and ubiquitinate and degrades p53.
So it makes sense to develop a small molecule inhibitor that will just cut off this vicious circle
and then we don't care that Hdm2 is present, you know, it can be there but it will be inhibited.
And that's exactly what happened.
Drug companies developed a drug, an inhibitor, the inhibitor is called Nutlin.
And again the beauty of it, that we are now, we are out of high throughput assays, screening libraries of zillion compounds,
it's a well designed drug.
It's well designed because the 3-dimensional structure of Hdm2 is known, in this case it's the mouse enzyme, the Mdm2.
And this is the pocket in which p53 is sitting.
You are designing a small molecule that occupies the pocket,
the pocket is being competed and p53 just cannot go there and it stays alive.
It's not ubiquitinated and not degraded.
And here is the experiment.
You can see that once we are adding Nutlin, p53 is going up,
the ligase is also going up because the ligase is a downstream gene to p53,
but we don't care that the ligase goes up because there is Nutlin in the medium.
So every molecule of Mdm2 that will be synthesised immediately will be inhibited by the drug.
And as a matter of fact, what you can see that P21 that is also down stream gene to p53, that is a cell cycle inhibitor,
is induced by the active p53.
And looking at facts analysis, you can see immediately that in non-treated cell, 45% of the cells are cycling,
they are synthesising DNA.
While following Nutlin treatment, 5% are in the cell cycle.
So we pushed 40% of the cells out of the cell cycle.
And here is a SCID mouse that was injected with tumour cells, with 1 million tumour cells.
And you can see that the tumour is growing in volume.
This is the tumour volume and here the tumour is treated, one treatment is Doxorubicin.
Doxorubicin is a highly toxic, non-specific chemotherapeutic agent, it's a DNA intercalator,
it's an oxyderivative of the nucleotide and it goes into DNA and it makes abnormal DNA.
And here you can see a very specific ubiquitin ligase inhibitor, Nutlin here,
the scores have the same effect as a broad spectrum highly toxic chemotherapeutic agent.
So if you go back to my story of the drugs, you can see the gradation, not the degradation, the gradation.
And the gradation is from non-specific treatment of the proteasome to more specific treatment of chaperones,
but it's still too broad because there are many proteins that are being capacitated,
that are being held by the chaperones to a very specific missile targeted treatment of ubiquitin ligase.
And this is the future.
Let me just end with something that I took from Craig, the signing,
but I want also to tell you there is a habit in the Nobel museum that laureates are signing chairs, the bottom of chairs,
but then not all of them sign the same chair from the same year, but they take the chair,
they let you sign and then they stash it for a few years and then they take it out again.
And it takes about several good years to fill the bottom of the chair.
So this is Richard Axel signing the chair and I took a picture.
But then I took a picture of the chair that I signed and, you know, I was so excited, I didn't notice.
So then I went back home and looked at it and I thought that I closed the cycle, a very nice cycle.
So that's me signed here.
Then I looked at the two other guys that signed it.
So one of them is a very close friend of mine that you heard here, and that's Tim Hunt, you see Tim Hunt.
And you see the cell cycle, you see the mitosis.
(Laughter).
This is the logo of Tim.
So you see the mitosis, you see the G1, the S way that we were just talking about,
with the Mdm2 and you see the G2 and here it says the cell cycle.
Now, why for me it's the closure of a cycle, not only that Tim is a dear friend,
but the cell cycle rides completely on the ubiquitin system because all the inhibitors and the accelerator,
the P21, P27, cyclin, cyclin dependent kinase inhibitors, Avram just showed you his wonderful work yesterday, is riding.
So it's going from the process to the mechanism, so it linked me back.
The other one that signed here is Ahmed Zewail, he's Egyptian by origin but working in Caltech, he's also laureate in chemistry.
And for me, to be on the same chair with an Egyptian, maybe it has some trajectory to the future.
Maybe by talking science we can talk also peace, thank you very much.
(Applause).
Thank you very much, that was a very nice talk.
So we have time for a few questions.
So Professor, thank you.
I would like to ask a question related to an interesting phenomenon in another system.
When we increase the activity of neurons,
we find that some special kind of proteins are selective degradated by ubiquitin proteasome system.
Why if we decrease the activity we got opposite results.
So my question is: In your opinion, how the ubiquitin protein system can be precisely regulated
for the degradation of a special kind of proteins, and why not others in the same cells? So thank you.
Ok, the question, if I understood it well,
has to do with the specificity of the system and how the ubiquitin system selects its certain substrate.
It has to do with the substrate itself in most cases, but also with the elements of the ubiquitin system.
So for example let's take nf-kappaB activation,
which is a very important transcription regulation that regulates the immune system
and is linked to chronic inflammation and then to cancer.
It's also a strong anti-apoptotic and it's activated by the ubiquitin system.
So following signalling, let's say by TNF alpha, you are phosphorylating very specific serine residues on the molecule,
on the inhibitor of the molecule.
On I kappa B alpha and I kappa B beta.
And these two specific phosphorylations that are set three aminoacids from one another,
so it's threonine 32 and threonine 36, will be specifically recognised by a very specific ubiquitin ligase.
So only this protein that has these two serines and these two serines were specifically phosphorylated by the specific kinase,
will be tagged at this moment, all other proteins will be spared.
And the same can go for p27, so the work of Michele Pagano and Avram.
So p27, which is a cell cycle inhibitor,
will be phosphorylated on threonine 187 and only following this phosphorylation a specific ubiquitin ligase called SKIP2
will come and take it away.
It doesn't mean that phosphorylation has just picked up two examples from phosphorylation
and how phosphorylation and signalling are feeding into the ubiquitin system, but there are many signals.
Take HIF-1 alpha, for example, in sensing of oxygen,
the wonderful work of Bill Kaelin from Harvard and Peter Ratcliffe from Oxford.
So normally, when we are in a normoxic environment, in high oxygen pressure,
we don't need to induce all these oxygen sensitive genes like VGF and VGF receptor in order to vascularise it,
because we have enough oxygen.
But only in low oxygen this VGF and VGF receptor endogenetic reaction is being stimulated.
How it's being regulated by a protein called HIF-1 alpha or hypoxia inducible factor,
it's a very potent transcriptional regulation.
Under hypoxia it's all the time oxidised.
And once it's oxidised on a specific proline residue, a specific ubiquitin ligase called the Von Hippel-Lindau protein will come
and take it away because it recognises the specific proline, hydroxyproline.
Under low oxygen there will be no hydroxylation, HIF-1 alpha will be up and will induce all the downstream gene.
So I just try to bring you two examples,
but the secret is always in specific recognition and therefore we need this huge repertoire of ubiquitin ligases.
Could you tell us about prion degradation.
Prion degradation, you should have Stanley Prusiner here to tell you about prion degradation.
Prion is extracellular, but it goes in and it has to do with the ubiquitin system.
I can just point out to a wonderful work that still is in the process and we still don't know,
and that is the secretion of the prion on its way out, that also somehow involves the proteasome,
and that's the work of Susan Lindquist from the Whitehead Institute in Boston.
And she claims that if you inhibit the proteasome, somehow you also accumulate the prion particle and you may induce even,
you know, the conversion of the PrPC into the PrPsc particle, and you may induce prion disease.
And then, you know, being sick is not good, but then you have to choose between the bad and the worse.
Do you want to die of prion disease or of multiple myeloma, I don't know, I mean it's a bad choice.
But we still don't understand obviously the full scope,
because the ubiquitin system is so complex and is involved in so many processes that we don't understand the sequel of it.
Thank you.