Good morning and as you heard from Avram in the previous lecture, I was his graduate student when we made the discovery
and Avram in his first point of lessons for life mentioned mentorship so maybe this is the ultimate proof
that mentorship is transmitted genetically.
So Avram had his grandparents, for me they are grandparents and I had my own excellent mentors, the first obviously was Avram.
And this was apprenticeship at its best because Avram as you can see here and as he mentioned works on the bench still.
But at that time our benches were just neighbouring to one another and we did experiments daily together
in an old monastery building that reminds anything else but laboratory.
So it was a truly fantastic period but then very similarly kind of following his steps also in our past education.
In many ways I went to a much bigger laboratory at MIT and I had another mentor, Harvey Lodish who is a fantastic biologist.
But again it was a very big laboratory and people were on their own.
But Harvey gave me the freedom to do whatever I want to do and I flip-flopped between degradation in the lysosome of ligands
taken to the cell by a receptor mediated entocytosis.
But then back to the ubiquitin system and we made further contribution in the ubiquitin field
when I was a post-doctoral fellow and from there on you're on your own.
And I think that one lesson is the following, yes even graduate student can make it at the end of the day.
But you really have to have really good mentors and to learn the lessons from them and I thought
that I was extremely lucky in that sense.
Ok what I want to tell you today is to take Avram's lecture one step forward and to show you the applied side
of the ubiquitin system and the applied side, it's not that we are translating it ourselves,
we are not doing any translational research, this was a little bit mentioned yesterday in the discussion.
But it just shows you that if you are doing a good research, at the end of the day there will be somebody
that will take it further and will apply it. And in our case its application to drug development and human diseases.
And what I want to show you today is this applied side of the ubiquitin proteolytic system.
So if we think about the ubiquitin system, so proteins are degraded and on the right side
you see that each protein is kept at a level and the level can be dynamic, it can fluctuate.
But if something wrong does happen, the protein can be excessively degraded and something,
it will go below its steady state level or there will be a mutation in a recognition site of a ligase
that was mentioned to you by Avram.
Or in the recognition motif of the substrate, it makes it associating with the ligase and then the protein will accumulate.
And both of these aberrations will lead to a disease and there are numerous diseases
that are attributed to either side of this equation.
Let it be either excessive degradation or under degradation of a protein.
And if we think about it, we can now look at it from a completely different side.
If we think of why it all will degrade protein, why we need to degrade protein and Avram mentioned it
but let's put it in a little bit of a different frame.
In each of the reasons of the main reasons why we degrade protein, there is a whole group of diseases that were described.
And obviously once you have a disease people are interested in the mechanism of the disease
and then they are making one step further and trying to go and develop drug.
So first of all it's quality control, there are many diseases that belong to quality control,
proteins are denatured, misfolded, aggregated, we need to remove them, if they are not removed properly they develop diseases
and I can bring you just 2 examples of big groups.
One group is obviously the neurodegenerative diseases that we discussed yesterday, partially in the master class,
Alzheimer, Parkinson, polyQ expansion diseases.
We have proteins aggregated in the brain, they are not properly removed and here there is a disease.
Don't be dragged behind me by this over simplification, not in all cases the proper degradation is the primary cause
but this aggregation and accumulation of protein always plays a pathogenetic role.
One disease that was mentioned beforehand is cystic fibroses.
Cystic fibroses is primarily a quality control disease where a mutated chloride transporter,
the cystic fibroses chloride channel is excessively degraded by the ubiquitin system.
Actually the mutated CFTR, the mutated protein is a normal functioning protein.
If you put it in a liposome it will pump chloride normally.
The problem is the mutation, the mutation is identified by the ubiquitin system.
And the ubiquitin system is there to control quality. And apparently the ubiquitin system fulfils its function.
It will remove it and the protein will not be expressed through the cell surface.
So here we have another kind of an excessive type of a quality control, where the system fulfils its function,
so just quality control one reason. Then control of processes, Avram brought you one of the ultimate examples, the cell cycle,
almost the entire cell cycle is riding over programmed and time degradation of different regulators.
And this allows the cell to move from one step of the cell cycle to another.
Wonderful work by Lee Hartwell, Tim Hunt, the word cyclin, it's called cyclin because it cycles.
And then the mechanism was unravelled by the further work of Avram or Kirschner and many others to show
that it's the ubiquitin system that controls it but not only this numerous oncogenes,
transcription factors are all degraded by the ubiquitin system. So we have to control processes.
Then obviously in order to maintain the differentiated and morphogenetic state of tissues
we have to remove proteins permanently and this is aberrated in many malignancies.
Now let's think about, I don't want just to bring you examples, I bring you only few highlights but I want to,
that we think conceptually, in the car we have 2 pedals, we have the gas and we have the break.
And in the pharma industry it has always been much easier to push, to put our leg on the break, to develop inhibitors.
You know think about cancer today, kinase inhibitors and so on. Namely to treat diseases of gain of function.
Once we have the processes accelerator, we are inhibiting the process by developing antagonist.
If we are losing function, if something is going wrong with an enzyme because it's mutated genetically,
if a function is lost it's much more difficult to replace it, we can replace it, you know hormonal therapy,
if you are losing insulin, you are treating with insulin in diabetes, adrenergic hormones, if we are losing them.
But it's much more difficult, think of Parkinson and think of other diseases, when we are losing function to replace function.
So the left side has always been easier and I think that the ubiquitin system is not an exception though
because the ubiquitin system is so complicated, we can manipulate it so that by developing inhibitors
we can still accelerate the system. And I'll show you later on how it works.
So this is one of the secrets of pharma or the hallmarks of pharma, the relative ease, nothing is easy, forget about ease,
in developing drugs for gain of function diseases, to develop inhibitors or to push the car break.
Compared to major obstacle developing drugs to loss of function diseases.
Accelerators and I brought you an example of genetic diseases, all the car gas pedal.
And the ubiquitin system has been converted in more than the recent decade for a large, large platform of drug development.
All the companies, basically all the big pharma and small pharma and start-up companies,
many of them are involved in the development and Avram mentioned one drug, I'll show you some other drugs,
I'll talk more about the drug that Avram mentioned. But this is just one review coming from natural reviews in drug discoveries.
And when you think about the ubiquitin system of where to drug it, you may think of the different hierarchy
and since the ubiquitin system, Avram didn't mention the number but the finite, or almost the infinite number of components
of ubiquitin system component is about 1,500, so there are about 1,500 enzymes and components in the ubiquitin system.
And if you think of the entire human genome, the entire human genome is about 22,000 open reading frames,
forgetting about splicing products.
So the ubiquitin system with all its tributaries is about 6, 7% of the entire human genome.
You can imagine that you have a large plethora of enzymes to be targeted.
From the first one, there is a single E1 that activates ubiquitin to about 30 E2's to about 100 of ubiquitin ligases.
And then to the proteasome and then all the modifying enzymes and so on.
So you can stratify the system and choose your target and accordingly and also conceptually, it's not just random selection
but rather you can do it in a very systematic way.
So there is a single E1, several E2's, hundreds of E3's and then thousands of substrates that are being ubiquitinated
and then funnelled into the proteasome to be degraded.
And indeed the ubiquitin system has been converted into a very large drug system.
We can stop polyubiquitination by UB statin. Avram mentioned Bortezomib or Velcade which inhibits the proteasome.
I shall talk later on inhibitors of recycling enzymes because at the end remember ubiquitin is a catalyst.
We mark the protein for degradation, the protein is being degraded but we retrieve back ubiquitin to go to the next target
and the next target. So ubiquitin is a covalent catalyst in many ways. So the enzymes that recycle ubiquitin are targets.
And then recently there is a whole family of ubiquitin-like proteins that were discovered and ubiquitin itself
serves many non-proteolytic functions, not only to target proteins for degradation and ubiquitin-like proteins
also serve numerous non-proteolytic functions.
They're called ubiquitin-like proteins because they are very similar to ubiquitin in structure
and also the mechanism of activation and conjugation is very similar.
One of them for example is SUMO, the other one is NED-8 and they conjugated to the protein in a very similar mechanism
to ubiquitin but it's only similar and not identical. They have their own conjugation machinery.
So as we saw in recent years the ubiquitin system has been expanded dramatically and ubiquitin that in my eyes
and in the eyes of many was a proteolytic signal was converted to become just another form,
very rich form however of cross translational modification.
One of the functions of which is obviously to target protein for downstream recognising protease which is the 26S proteasome.
So you can see that the ubiquitin system has become into a very rich drug development system and let's start with cancer.
And Avram already pointed out and let me again wrap it up in an over simplified picture, that if a tumour suppressor like P53
is excessively degraded or an oncogenic protein, a growth promoting factor is inhibited from being degraded
we are up into a cancer.
And we can think of P53 on the right side of the equation, let's take human uterine cervical carcinoma,
you heard about it from Harald zur Hausen, he is kind of the father of the field, the human papillomavirus encodes
an oncogene called E6, E6 heterodymerise to P53, this is a purposeful process evolved by the virus.
This heterodimer is somehow recognised by the ubiquitin system as a foreign protein
and the ubiquitin system trashes the tumour suppressors to the sink, to the proteasome.
The virus does it purposefully because P53 is a quality check point protein.
And if it will be there it will prevent the virus from integrating into the cellular DNA and from executing its numerous function.
So the virus was very smart, he smarted out the ubiquitin system by evolving an early expressing gene
that will target first the tumour suppressor for degradation. Allowing then the virus to go ahead and being expressed in the cell.
So here we are trashing a tumour suppressor protein that is there to check quality, to check the integrity of the genome
and afterwards the virus is free. And at the end of the process we are seeing malignant transformation.
On the other side we can look at the EGF receptor that from time to time is promoted or to beta catenin for example
that from time to time is not degraded.
And it keeps on signalling to the cell and the cell keeps on thinking that it's under constant stimulation for division
and for expression of cell division proteins and it keeps on running in an uncontrolled manner because the regulator,
either beta catenin or the EGF receptor, and I can bring you numerous other examples,
is not under degradation control anymore of the ubiquitin system.
So you see deviation in any side of the ubiquitin system will lead to aberration and one of the first drug
was surprisingly developed, as was mentioned by Avram as a proteasome inhibitor, it's an inhibitor of specific site,
of the active site in the proteasome.
The proteasome is, you are going to heat it from Robert Huber later on, from 2 sub-complexes,
this is the proteolytic arm of the system that degrades ubiquitined proteins.
And here are the active sites in the middle, they are sitting here in the catalytic chamber, in the catalytic particle.
And here you can see them, these are threonine residues and they are inhibited specifically by Velcade
that was shown to you by Avram, it's a tripeptide, a boronic acid derivative
and it attacks electrophonically specifically the threonine residue to inhibit it.
And here you see a patient, this is a patient with multiple myeloma, multiple myeloma for those of you
who are not in the medical field is a monoclonal expansion of a B cell, of an immune IG secretory B cell in the bone marrow.
It occupies the entire space of the bone marrow.
Suppresses all other progenitors and the patient is suffering heavily from this occupation of space.
Tumours are space occupying lesions if we want to think about it.
And in this case it's the suppression of the other elements so the patient is suffering from lack of erythrocyte,
difficulties in breathing, lack of leukocytes, infections, bleeding because of lack of thrombocytes
and then pathological fracture because the tumour presses against the vertebra and against the lung bones.
And as Avram mentioned this disease has been lethal in suffering until several years ago when 2 drugs came into the market,
one of them is Velcade, the other one is a derivative of thalidomide, I'm not going to talk about it.
And here you can see a patient, you can see the galloping, the riding up level of the immunoglobulin secreted by the tumour.
So in this case we have an excellent biomarker, treatment starts here, chemotherapy, you see partial remission
but then relapse again and then treatment with Velcade started and you can see a depression of the bio marker.
Not all patient respond, we are now not we, researchers are profiling patients to sort out, to personalise medicine,
to tailor the treatment to the patient because not all patients respond
and we are interested to know why certain do and why certain do not.
And here you can see the bone marrow of the patients and you can see here the malignant homogenous bone marrow
scattered with these B cells and following treatment you can see that the bone marrow is rejuvenated,
being refilled again with the normal progenitors.
Here you see a sister disease, this is a CAT scan of the chest, it's a non-Hodgkin lymphoma, it's kind of a cousin disease
and you can see here this is a cross section of the chest for those of you who are not oriented anatomically.
So the CAT scan is a wonderful technology, also was awarded the Nobel Prize.
And what you can see here, we cut the chest and we kind of lift and we look inside but we do it metaphorically.
So here you can see the sternum, the front chest bone, you can see nicely the vertebra with the spinal canal,
you can see the scapula, the bone of the shoulder, the shoulder masses, the ribs and you can see here
a tumour sitting in the hilum, in the gate of the right lung.
The black is obviously the air in the lungs, here you can see the great vessels of the heart and here the tumour regressed.
So you can see a nice treatment. Let's go to another layer. So forget about proteasome inhibitor, let's move further.
Chaperones, chaperones are quality control proteins that their function is to refold proteins and to put them back into the pool.
So before you go ahead and degrade a protein, you still give the protein a chance to be refolded.
But from time to time in the language of Susan Lindquist who is a very outstanding chaperone researcher, chaperones becomes,
here you can see it on the left side, evolutionary capacitates of mutation.
So they hold on to the mutated proteins and they don't let it out, they don't refold it,
but they don't subject it to the ubiquitin system.
So here the protein has found itself protected from degradation and keeps on executing its mutated aberrant function.
So the idea will be to dissociate this Gordian knot between the chaperone and the mutated proteins.
And different inhibitors, different dissociators were evolved and one of them is geldanamycin,
it turned out to be toxic but here you have a florescent staining of breast cell, culture cells with the EGF receptor,
with the mutated EGF receptor.
And following treatment with the dissociator you can see that the florescence is clear, the protein was released
and went down following ubiquitination to the lysosome. And here you can see the biology, I mean it's beautiful, the chemistry.
So this is a different dissociator, you can see that the protein,
the ErbB2 which is a derivative of the EGF receptor following addition of the dissociator is getting ubiquitinated.
The protein itself disappears but mostly all those growth promoting factors are self-stimulated kinases.
So one day ligand binds to it, they go and kinase themselves and then they are going to be activated
for their further downstream activity.
You see that the kinase activity, the phosphotyrosine, this is a specific antibody to phosphotyrosine,
on the receptor itself is completely gone.
And we can look at the patient with multiple squamous cell carcinomas in the face,
you can see that following 47 days this big tumour is now ulcerated so it's on the way to healing and the face are better.
And following additional 40, almost 40 days, the face are much better. So we are taking a mechanism and converting it into a drug.
But the next generation of drugs is going to be inhibitors of ubiquitin ligases because the plethora there is very rich
but it turned out to be a rock hard to crack. Why?
Because ligases interact with a proteolytic substance of ubiquitin system in a low affinity on large surfaces.
And it turned out to be very difficult to tamper with.
So therefore the field is a little bit behind in developing inhibitors to ligases but nevertheless people are attempting
and I'll shop you one attempt. And this is P53 again.
I am coming back to this critically important tumour suppressor or check point, quality control protein.
And this protein is really jumping up in any stress, you stress the cell, you just touch the cell,
telomere shortening, ribonucleotide depletion, mitotic spindle damage, DNA damage, any damage, P53 will jump up.
And once it jumps up it will immediately stop division of the cell.
Because the idea is that the cell with the damage cannot proceed, we want to stop it from being dividing.
Then there will be an attempt on damage repair.
So downstream to P53 there will be a whole host of repair enzymes and genes that are activated, whether it's in the DNA
or in other damage oriented directions.
And if after a trial and it's going along a time line, the damage cannot be repaired, then P53 will induce apoptosis.
It will kill the cell and take it out of the cellular repertoire.
So P53 is critically important protein and just to show you how critical it is, mutations in P53 were shown
to be part of about 50% of all known tumours, it's a critically important protein.
And in another 50% of the remaining 50%, so we are talking now 75%, P53 is down regulated, it's wild type, it's not mutated,
it's functional but nevertheless it's down regulated. How it's down regulated by its own ubiquitin ligase.
So here the ubiquitin system plays a pathogenetic role.
It takes a tumour suppressor and down regulates it in order to allow cancer to proceed.
How it down regulates it by up regulating its ubiquitin ligase.
Normally the ubiquitin ligase of P53 which in mouse is called MDM2, in human it's called HDM2,
is in a servo loop regulatory mechanism along with P53. So P53 is a transcription factor, it up regulates its own executioner.
The executioner ubiquitinates and degrades P53, P53 is down, once P53 is down it cannot transcribe anymore the ligase,
then the ligase is down but then P53 is up. And then the story goes on and on and on.
So they cycle in a sinusoidal phase difference from one another and this has been shown beautifully.
In those 25% of tumours in which P53 is of the wild type, HDM2 goes up in an uncontrolled manner,
it doesn't listen anymore to the regulatory voice of P53, it's going out of control.
It's constantly up and it turns down, it down regulates P53 all the time.
so here you have a mechanism, what shall we do, we shall develop an inhibitor to MDM2,
if we shall use a small molecule inhibitor to HDM2, HDM2 will be there, it will be high but it won't be able
to recognise P53 anymore and P53 will go up again. Let's see how it works.
It does work, there is an inhibitor called Nutlin, was developed in Nutlin New Jersey by Roche,
it's still an experimental inhibitor and the inhibitor is based again on understanding of the 3-dimensional structure
of the ligase and it's a small molecule that sits exactly where P53 is sitting.
So it competes out, P53 out of the active site of the ligase.
And let's look at the result of the experiment, here it is. We are adding the inhibitor, P53 is coming up - fantastic.
So here is the inhibitor.
MDM2 is also coming up, so the executioner is coming up but we don't care because the executioner is dead
because there is an inhibitor in the medium, here it is.
So we get, P53 is active, it transcribes its executioner but the executioner is dead because there is an inhibitor
that is all the time there.
And how do we know that the P53 is a good one, because P53 now induces P21 and P21 is a cell cycle inhibitor.
And how do we know that we inhibit cell cycle, we look at the facts analysis.
In the facts analysis in the control almost 50% of the cells are synthesising the DNA. They are cycling.
While afterwards once we are adding the inhibitor only 5% are there. So we are pushing the cell out of the cell cycle.
And here is the result in mice, in SCID mice, you can see we are injecting tumour cells into the back of SCID mouse
and the tumour is growing, what you see on the ordinate is the tumour volume and you see that it's growing.
And once we are injecting Nutlin you can see that the tumour stopped from growing and the effect of Nutlin
is identical to the effect of Doxorubicin which is highly cytotoxic DNA chelating agent.
So with a specific inhibitor of ubiquitin ligase we can achieve exactly the same result as we are achieving
with a more broad highly toxic chemotherapeutic agent. So this is a good sign for the future.
As time closes up I just want to go to the very last slides and to show you how the field really evolved,
this is just a paper that I found out in last Cell, Cell is very famous for those of you who are in biology,
very famous periodical, came out last Friday.
So it just shows you how rapidly the field, in this case they are targeting E2 and this is the E2
that is involved in cell cycle, completely allosteric, new inhibitor that just came out, just to show you how dynamic it is.
But I want to know, and then there are inhibitors of ubiquitin-like proteins that I don't want to tell you
but I want to go to the very last 2 slides.
And to show you how we can manipulate the ubiquitin system even to loss of function diseases and this is very interesting.
And let's go to neurodegeneration.
And in neurodegeneration as I told you we have aggregated proteins and this is loss of function and we are standing hopelessly.
So the story is the following: We have the proteasome here, we have the proteasome and the proteasome degrades ubiquitin
into proteins and the proteasome is equipped also with deubiquitinating enzymes to recycle the ubiquitin.
So a protein is approaching the proteasome, the protein is degraded, ubiquitin is recycled.
If we shall manipulate a little bit the proteasome and inhibit partially the deubiquitinating enzymes
we shall make degradation a little bit more efficient.
So we can manipulate the 2 processes because the proteasome is a dual function enzyme.
So an inhibitor was developed by Dan Finley at Harvard medical school, this is the nature paper that showed up a few weeks ago
and I just want to show you one last panel and this is this panel. The C panel.
And you can see here that once we are inhibiting the inhibitor an experimental protein that is called Q80 or Q22 is going down.
Now what is Q80 or Q22, I mention to you one degenerative disease which is Huntington disease.
In this group of diseases which is not only Huntington but also spinocerebellar ataxia and Kennedy's disease
and many other diseases, there is a genomic instability and a protein is expending its N terminals by repeating
in cooperation of glutamine so there is a CAG repeat which is the coding triplet for glutamine,
typically we have very few of them and all of a sudden we get an expansion on certain proteins.
And we get a protein that is a normal protein but it's preceded at the N terminals by a large stretch of polyQ's
and these polyQ's are aggregated, precipitated and lead to this aggregated result which is the disease,
let it be Huntington, spinocerebellar ataxia and so on.
And here you have a model in which we are manipulating one activity of the proteasome, the deubiquitinating enzyme,
pushing the proteasome to be a little bit more efficient and are able to get rid of an aggregated protein.
So here we are starting already to move from diseases of loss of function to diseases of gain of function
by manipulating activity and by having a very rich plethora of the ubiquitin system.
So I think that I, I hope that I gave you a little bit of a flavour of what started in the late '70's of me
becoming a graduate student of Avram, just curious about a small unclosed niche in biology,
ending up in drugs and more than that in a big future for drugs. So thank you very much.