We're sorry but this page doesn't work properly without JavaScript enabled. Please enable it to continue.
Feedback

The Proteasome, Structure, Mechanism, Ligand-Binding, and Application in Drug Design and Development

00:00

Formal Metadata

Title
The Proteasome, Structure, Mechanism, Ligand-Binding, and Application in Drug Design and Development
Title of Series
Number of Parts
340
Author
License
CC Attribution - NonCommercial - NoDerivatives 4.0 International:
You are free to use, copy, distribute and transmit the work or content in unchanged form for any legal and non-commercial purpose as long as the work is attributed to the author in the manner specified by the author or licensor.
Identifiers
Publisher
Release Date
Language

Content Metadata

Subject Area
Genre
Abstract
The proteasome plays a key role in archaeal and eukaryotic cells by proteolysis of incorrectly folded, non-functional proteins (in archaea and eukaryotes), and of ubiquitin-labelled signalling proteins (in eukaryotes) and antigenic proteins (in vertebrates with an adaptive immune system). The eukaryotic proteasome is composed of two components, the regulatory cap and the core particle (CP), which harbours the active sites. Its activity is controlled by sequestration of the substrate-binding active sites inside the barrel-shaped architecture of the CP equipped with entry gates at both ends, which are closed in the latent state and opened induced by cofactors leading to activity. My presentation focuses on CP which has been extensively characterized in structure by x-ray crystallography (Groll, M., Ditzel, L., Löwe, J., Stock, D., Bochtler, M., Bartunik, H. D. and Huber, R. (1997). "Structure of 20S proteasome from yeast at 2.4 Å resolution." Nature 386, 463-471.) and by in vitro and in vivo functional studies. It consists of 28 subunits arranged in 4 stacked hetero-heptameric rings, alpha1-7, beta1-7, beta1-7, alpha1-7. The assembly of CP commences sequentially with the formation of the alpha rings onto which beta subunits affix in a defined order and is followed by autolytic cleavage of the N-terminal pro-peptides thus exposing the N-terminal Thr residues of three of the seven beta subunits (1,2,5), which are enzymatically active. (Groll, M., Heinemeyer, W., Jäger, S., Ullrich, T., Bochtler, M., Wolf, D. H. and Huber, R. (1999. "The catalytic sites of 20S proteasomes and their role in subunit maturation: A mutational and crystallographic study." Proc. Natl. Acad. Sci. USA 96, 10976-10983.) Access of the active sites for substrate requires opening of the entry gates formed by the entangled N-terminal segments of the seven alpha units. Mutation of a single residue in alpha3 generates a constitutively active enzyme. (Groll, M., Bajorek, M., Köhler, A., Moroder, L., Rubin, D. M., Huber, R., Glickman, M. H. and Finley, D. (2000). "A gated channel into the proteasome core particle." Nature Struct. Biol. 7, 1062-1067.) The enzyme mechanism proceeds via formation of a tetrahedral adduct of the threonine hydroxyl group with the carbonyl carbon of the scissile peptide assisted by invariant lysine33 and aspartate17 residues, continues with acyl enzyme formation and peptide bond cleavage, and finishes by water mediated ester hydrolysis and liberation of the products. The majority of the specific ligands discovered and developed for the proteasome target the threonine nucleophile by aldehyde-, lactone-, boronic acid-, epoxyketone-, unsaturated amide- head groups (war heads) covalently. Noncovalent ligands of different chemistries have also been found. Natural compound libraries appear to be a rich source. (Groll, M., Huber, R. and Moroder, L. (2009). "The persisting challenge of selective and specific proteasome inhibition." Journal of Peptide Science 15, 58-66.) Research in proteasome inhibitors is strongly encouraged by the discovery of the proteasome as a drug target for leukemic cells and haematological tumors. Three proteasome inhibitors have been developed, are clinically applied, and commercially very successful. Vertebrates have three different CPs. The constitutive proteasome (cCP) is ubiquitous in all tissues, whereas the immunoproteasome (iCP) is found predominantly in lymphocytes. cCP and iCP differ by a unique set of catalytic subunits beta1,2,5 displaying modified substrate binding pockets and enzymatic specificities and activities. Selective inhibitors of the immunoproteasome have gained interest offering new strategies for autoimmune disorders, inflammation, rheumatoid arthritis, and certain types of cancer, and promise/show reduced side effects such as peripheral neuropathy. (Arciniega, M., Beck, P., Lange, O.F., Groll, M. and Huber, R. (2014). "Differential global structural changes in the core particle of yeast and mouse proteasome induced by ligand binding." PNAS vol.111 no.26, 9479-9484; Huber, E.M. and Groll, M. (2012) “Inhibitors for the immune- and constitutive proteasome: current and future trends in drug development” Angew.Chem. Int. Ed. 51, 8708-8720; Cromm, P.M. and Crews, C.M. (2017). “The proteasome in modern drug discovery: second life of a highly valuable drug target”, ACS Cent. Sci. 3(8), 830-838.) Various lines of development based on different chemical scaffolds are pursued in Academia and Industry (www.Proteros.com; www.lead-discovery.com) guided by high resolution crystal structures and give hope for new therapeutic options for diseases with high medical need.
Binding energyNobeliumLigandRecreational drug useCell (biology)ArzneimittelforschungCancerNobeliumPhotosynthesisChemical reactionProteinMultiprotein complexChemotherapyChemical structureOrganic chemistryLecture/Conference
PelletizingReaction mechanismLigandArzneimitteldesignBinding energyNobeliumProteinfaltungChemistryArzneimittelforschungCell (biology)Pharmaceutical drugMultiprotein complexProtein biosynthesisErdrutschWine tasting descriptorsSystemic therapyProteinMeeting/InterviewLecture/Conference
ProteinProteaseProteinCytosolAcidAmineBiosynthesisPeptideGenregulationInhibitorEnzymeTofuAngular milMill (grinding)Metabolic pathwayUbiquitinPlatinThin filmMantle (geology)ProteolysisChemistryBiochemistryEnzymeEukaryotePharmaceutical drugHydrophobic effectProteinTrace elementChain (unit)UbiquitinProteaseProteolysisBaker's yeastIsotopenmarkierungActivity (UML)AcetoneKorkenNanoparticleBlock (periodic table)ProteolyseChemical reactionOrganic chemistrySeleniteReaction mechanismX-ray crystallographySystemic therapyHydrolysatActive siteAmino acidStereoselektive SyntheseTreibsatzAle MunicipalityElectronic cigaretteWaterfallCrystallographyMoleculeFireErdrutschLeft-wing politicsWine tasting descriptorsChemical propertyProtein subunitGeneElectronCell (biology)Computer animation
TiermodellBaker's yeastActivation energyGesundheitsstörungAssembly (demo party)GenregulationProtein subunitInsulinInhibitorReaction mechanismAssembly (demo party)Quantum entanglementProtein subunitRecreational drug useHydrophobic effectN-terminusAspartic acidProteolyseActive siteEnzymeMutageneseWine tasting descriptorsNanoparticleInhibitorNucleosomeBaker's yeastActivity (UML)Reaction mechanismElectronAtomic numberPeptideSunscreenPotenz <Homöopathie>MalerfarbeStockfishErdrutschGesundheitsstörungFunctional groupVancomycinAbbruchreaktionHeck-ReaktionGenotypeMan pagePainDiseaseHuman subject researchPitch (resin)Dietary supplementGezeitenküsteExplosionTeaComputer animation
NobeliumReaction mechanismFunctional groupAageGenetic disorderReaction mechanismOrganic chemistryLecture/Conference
BioavailabilityReaction mechanismActive siteProteolysisInhibitorMultiprotein complexSymptomSeedlingSunscreenAgarPeptideStereoselectivityChemistryParasitismTuberculosisDiseaseBinding energyEnzyme inhibitorReaction mechanismPaste (rheology)AbbruchreaktionDiseaseSerineLactoneChemistryFunctional groupMoleculeChemical plantParasitismCyclineRecreational drug useProteolyseEnzyme inhibitorBinding energyChemistHydroxylCancerWursthülleWaterEnzymeNaturstoffThermoformingN-terminusAcylInhibitorPeptideChemical reactionSinger CorporationPotenz <Homöopathie>AntigenAreaPathogenicityTrauma (medicine)Sense DistrictCrystallographyStratotypGolgi apparatusAageAssimilation (biology)OrlistatAtomic numberActive siteLecture/ConferenceMeeting/InterviewComputer animation
NobeliumPhysiologyFunctional groupLactitolReaction mechanismLecture/Conference
Cell (biology)Functional groupPhysiologyWine tasting descriptorsMeeting/Interview
NobeliumInhibitorBortezomibWasserbeständigkeitMajor histocompatibility complexMoleculeAntigen-ProzessierungResidue (chemistry)ToxicityConstitutive equationBase (chemistry)CancerPathologyGasOncogeneDiseaseCell (biology)Functional groupCell cycleKlinische ChemieSymptomProtein subunitTiermodellSystemic therapyThylakoidAlumPharmaceutical drugProteaseTeilentrahmte MilchMeat analogueRecreational drug useReaction mechanismStructural elucidationLeadChronic (medicine)Sample (material)Covalent bondEnzyme inhibitorCollagenImmunologyTargeted drug deliveryFunctional groupSense DistrictToxicityPolymorphism (biology)ErdrutschAreaCell (biology)DiseaseFermentation starterHope, ArkansasDeterrence (legal)CheesePlasticGesundheitsstörungMultiple myelomaWhite blood cellPharmaceutical drugEnzyme inhibitorChemical elementWine tasting descriptorsBlock (periodic table)WalkingTool steelRock (geology)Curing (food preservation)NeuropathologieSingulettzustandRheumatismPotassiumInhibitorAmino acidHematopoietic stem cellStereoselectivityLeadRecreational drug useThreonineStimulus (physiology)ProteinTumorArzneimittelforschungProtein subunitActive siteLecture/Conference
NobeliumErdrutschRecreational drug useBase (chemistry)Lecture/ConferenceMeeting/Interview
NobeliumProtein subunitInhibitorEnzyme inhibitorLecture/Conference
InhibitorMoleculeGenotypeGesundheitsstörungEnzyme inhibitorMeeting/Interview
NobeliumExplosionProteaseResistenzGesundheitsstörungProtein subunitCell (biology)CancerBortezomibLecture/Conference
ArginineTumorAcidDiseaseSpaltflächeInhibitorBortezomibEmission spectrumDeterrence (legal)Constitutive equationLeadEnzyme inhibitorNeotenyProtein subunitChemical compoundPotenz <Homöopathie>CigaretteHuman subject researchProteinLecture/ConferenceMeeting/Interview
NobeliumChemical reactionNobeliumPhotosynthesisProteinAprotininFunctional groupProteasePharmaceutical drugKristallkörperLecture/ConferenceMeeting/Interview
NobeliumRapidTool steelCrystallographyProteinX-ray crystallographyElectronic cigaretteChemistPharmaceutical industryRecreational drug useMoleculeLeadLecture/ConferenceMeeting/Interview
NobeliumComputer animation
Transcript: English(auto-generated)
My name is Melissa Follett. I'm a science journalist based in Montana, and we are here to talk about the proteasome and its role in cancer development,
cancer drug discovery. I'm here with Professor Robert Huber. He won the Nobel Prize in 1988 for contributing to the structural determination of the protein reaction center that's key to harvesting light energy in photosynthetic organisms.
And what we're going to talk about today is this really key protein complex in cells. It's got such a key role in cells that it's actually a really unusual target for drug discovery. But nevertheless, the structure of this complex has helped develop drugs
that are now being sold for billions of dollars each year. So, Professor Huber. Well, good morning. Thank you. Now, this is a slide that shows the life cycle of proteins,
from birth protein synthesis to maturation and to death. Now, we know a lot of details about these different mechanisms, and some of them have been presented and are being presented at the Lindau Conference.
Now, my story is about protein degradation performed by proteases, which is in principle a very simple chemical reaction hydrolysis of a peptide bond.
However, it turns out that in higher organisms we have more than 600 different genes for proteases. So why is this so? It has to do with the fact that limited proteolysis is a major regulatory mechanism by activating or deactivating proteins and enzymes.
So, on the other hand, proteolytic activity is quite dangerous. It could kill cells, could kill organisms if left uncontrolled. And what I have been doing during more than four decades of research
was looking at the different regulatory mechanisms that we find in nature. And the astounding fact was that there are extremely diverse regulatory mechanisms.
Unfortunately, the pointer doesn't really work. So, what we found is a very simple mechanism, which you see on top of this slide, where a red protein interacts tightly with the green protease and blocks excess of the substrate.
So these interactions can be very specific, very tight, and much stronger than a substrate. So, quite clearly, substrate proteolytic activity is inhibited. Now, we can go through the list. No time for that.
Now, I come to the last mechanism that we found, which leads me then to the proteasome. That is the mechanism whereby the active sites are inside a particle not accessible from outside
because there are entry gates, which are closed, and then we have the latent, the inactive form, or opened, and the regulatory mechanism is opening and closing of the entry gate.
This is the story of the proteasome I would like to tell you today. Now, the proteasome is the major intracellular protease. It is related to the ubiquitin system. You have heard and know about the ubiquitin system.
There were lectures here about it, whereby ubiquitin labels a protein that is to be degraded. Then this labeled protein is targeted to the proteasome, whereby a complex reaction, the label is removed,
ubiquitin is released, the target protein is unfolded so that it can then be thread into the particle where it is digested.
This is the basic mechanism of the proteasome. This is the basic structure. There is a central component, the so-called 20S proteasome, in which the proteolysis is carried out,
and it has regulatory caps which deal with the recognition and the removal of the label, the polyubiquitin. Most of the structural studies have been done on the core particle,
and this is also that component for which drugs have been developed, and also this is part of my story. This is the timeline, the milestones in structural biology of the 20S proteasome.
It started with relatively diffuse electron microscopic pictures of negatively stained preparations of the 20S proteasome, which you see on the left-hand side, which have been improved. Ten years later, to have a more detailed picture, but again, of course, far from atomic resolution,
negative stain preparation, 1988, and then the breakthrough with the first crystal structures. First, the archaeal proteasome, and then the eukaryotic yeast proteasome, and then later, mammalian proteasome.
I should mention that the authors of the archaeal and the yeast proteasome were Jan Loeb and Michael Grohl. Now, Jan is director of the LMB in Cambridge, a really prominent position.
Grohl is professor of biochemistry at the Elite University, Technical University in Munich. So obviously, work on the proteasome supports an academic career very much, and still does, I think. These are the chain tracings of first the archaeal proteasome and then the yeast proteasome in comparison.
So it consists of 28 subunits, a huge molecule, and it was at the time when we published in 1995
the largest asymmetrical protein molecule analyzed by X-ray crystallography. Now, you see that the archaeal enzyme and the eukaryotic enzymes have the same architecture.
Both consist of 28 subunits, but the chemistry is very different. That is, the archaeal enzyme consists of just two subunits, 14-fold, repeated in the structure. It has a seven-fold symmetry, so it has an alpha ring, which is a homoheptamer,
and then comes a beta ring, a middle ring, which carries the active site. I'll come back to that later. A homoheptamer, while the yeast, the mammalian, the eukaryotic proteasome, has seven different alphas.
So it's a wonderful example of how evolution works. So it clearly began with the symmetrical archaeal enzyme and then evolved by making the subunits different, more specific, but I'll come back to that later also.
Now, the essential point I want to show here is that the active site, that is where proteolysis happens, is inside the part, again, associated with the beta subunits and associated, as you will see,
with the N-terminal trionin. That becomes important when we think about inhibitor design and drug design. Now, time has progressed, and we see right now this revolution in structural biology by cryo-electron microscopy.
Now, this is the first cryo-EM analysis of the holoproteasome. It is the core particle plus the regulatory particles, which I showed you before. Beautiful work.
Of course, it's not atomic resolution, but it's close to it. And because we know so much about the detailed structure of the individual components, one can fit these into the relatively low-resolution EM map.
So clearly a revolution in structural biology, electro-microscopy. You had lectures in that field during the Lindau conference. Well, a question, of course, is this is a particle, the core particle of 28 subunits,
how does it assemble such that each subunit knows exactly its place in this arrangement of four rings, heptameric rings, that we have studied. And it turns out that the assembly begins with a heptameric ring of the alpha subunits.
So they form a stable crystallizable ring. Now, this is then the template for the beta subunits, to which they attach to form an inactive half core particle.
Now, essential for the assembly of the beta subunits is the presence of propeptides that they have. I told you that the activity of the proteasome depends on a free N-terminal trionin.
Now, but all the beta subunits are synthesized with a propart, which must be cleaved off. And that happens when two half particles associate, then activity develops such that the active site trionin then attacks its preceding peptide bond,
which is a glycine, and performs proteolysis. And then this propeptide is then cleaved off, the free N-terminus is liberated,
and we come to the active core particle, which is still latent. Now, why is it latent? So it has very low activity.
It's latent because the alpha subunits, which form the outer rings, have a tight entanglement of their N-termini. So this is the closed door situation, which I mentioned on the lower left-hand side of the slide.
You see the electron density in the middle, so showing a tightly packed structure. It's very irregular because these are seven different alpha subunits, very irregular, but forming a well-defined structure. Now we can do mutagenesis in yeast easily there,
and pick out one single non-covalent interaction, which seemed to us to be essential for the structuring of this entry port.
So we make a single mutant. The interaction is led by an aspartate with the tyrosine, which sits in the middle of this plaque, and we mutate the aspartate to an aspartate.
This means it's just a single-atom mutation, which has a profound change, makes a profound change to the structure. Now you see here that the structure of this entry port, which is well-defined in the wild type, completely disappears.
You do not see any electron density, but the matter is there. It's just not ordered, and at the same time, the activity shoots up by a factor of 100. So a single-atom change makes a profound alteration of the structure and the activity.
So this may be important for the proteasome in neurodegenerative diseases, where one believes that the proteasome is not properly functioning.
But this is another story. Well, I mean, we could invite questions to that part, and then I come to mechanism and inhibition. There are questions.
You have one. So I had a quick question about the correlation of proteasome function and aging. In several organisms, I believe there is a programmed decline in proteasome activity,
which is also why neurodegenerative diseases happen at a later stage in life. Can you explain the mechanisms of this correlation, of this decline?
I don't think I can. It may have to do with less proteasome around. It may have to do with what some people believe, that the proteasome is overwhelmed by incorrectly folded proteins,
and that does not properly work. So these are theories. I don't know. We will certainly have an answer in the future. So about mechanism. Now, I told you that the proteasome is an N-terminal trionine protease,
the first one that was discovered when we first, when we revealed the crystal structure of it. So it has an N-terminal trionine, which then attacks the hydroxyl group as a nucleophile, attacks the sisal peptide bond, forms a tetrahedral intermediate, which then is cleaved,
and one of the products is released, forming an acyl enzyme that is in the lower part on the right-hand side,
and then there is de-acylation with an incoming water molecule. So a very simple chemical reaction that we see in most of the proteases, in particular the serine proteases, where it is a serine that is the nucleophile, here is a trionine, which is the nucleophile.
Now, the fact, and I will come to that later, that the proteasome was found to be a cancer target, a normal strategy for fighting a cancer,
caused many people to screen their libraries, chemical libraries, but in particular the natural compound libraries, for anti-proteasomal activity. And the first one was discovered when we worked on the Arhael proteasome.
This is called omorolite, and it is a lactone that oscillates the N-terminal trionine, and of course inhibits the enzymatic function. This is the kind of experiment that we do by crystallography.
It's relatively high resolution. We see clearly defined each of the atoms, showing that there is an oscillation of the N-terminal trionine. This is, well, many more we have looked at, because people discovered new proteasome inhibitors
in natural compound libraries. They sent them to us to define their binding. Now, we were involved in some of these discoveries. This is a plant patuchen, a Pseudomonas syringae, which needs a virulence factor,
which is shown here, syringeline A, and we found out what it does. It binds to the plant, in this case bean plant proteasome, and inhibits the proteolysis of cyclin B1.
This you see because you find a ladder of polyubiquitinated cyclin B1 that then leads to apoptosis, so a very clear mechanism. Of course, this molecule has then been synthesized with an attempt to develop it
into a human drug as well, ongoing work in a number of places. Well, an interesting aspect of this syringeline of Pseudomonas
was that human patuchen, Burkholderia, Pseudomonale, patuchen also needs, which causes, sorry, which causes parasitic pneumonia,
which causes this disease, needs virulence factor, which is called ketobactinin, which has the same chemistry as the Pseudomonas plant patuchen. Again, this has been synthesized with an attempt to continue in drug development.
Well, another aspect of proteasome research was the finding of American colleagues, though the original citations you find here, that there are proteasomes in the malaria parasite, in plasmodium,
in other parasites that are causing Leishmaniasis and Chagas disease, sleeping sickness. So they have their special proteasome, and one can analyze these,
even structurally define, and search for small molecules that are specific, can be found, but do not touch the human proteasome. They are not toxic. So there is a lot of development in the antibiotic field with the proteasome
as a target, extremely interesting development. Now this is the... We'll pause here and ask for questions. Yes, of course. Okay. Any questions? To this point? We have about, there's back there. Yes.
I have a question about the first part. I'm sorry that I was late to question, but you mentioned that the 20S itself has much less activity compared to 26S, but the activity is not zero.
Do you think there is a physiological importance in 20S mechanism? Because there are some papers indicating the importance, but there is no great method to assess the function of 20S itself. Now it's well known that 20S is quite abundant in cells.
So probably even more abundant than the 26S. So it has a function, and it has some low activity which may be stimulated by certain substrates as well. So it is there. It has an independent... Well, not an independent, but it has its own function
and is clearly relevant in human physiology. I think I saw maybe one. Was there another question? I think I saw. Okay.
Well, as I told you, the proteasome has, to great surprise, at least from my side, become a very important drug target. Why surprise? Because of the essential function that the proteasome has.
So how can that be a target? Now, obviously, when we had published the first structure, of course it was open, available, and people started to think about developing inhibitor and, to a surprise, found that proteasome inhibition
is a new strategy against multiple myeloma, an entirely novel strategy, so very different from the usual strategy, which are mostly based on kinases.
So the interest was great. It was a successful new drug, and as you see from these numbers there, big selling. So that again stimulated then the search for second- and third-generation proteasome inhibitors.
Again, these were successful. Some are on the market. Of course, all of them have been designed and developed on the basis of the publicly available crystal structure
that you may regard as a nuisance, but I did not. I was happy to have helped in the development of these new drugs. Now, the story goes on because these drugs
against the constitutive proteasome, the proteasome is in all cells. The constitutive proteasome is a drug target, but it shows very severe neuropathic toxicity,
so severe that there are quite a substantial number of patients that receive treatment with proteasome inhibitor, in particular with this well-known borthesome inhibitor
that has to be stopped because of this serious neuropathy. Now, it was known that in immune cells there is a variant of the proteasome,
a variant in that sense that the sum of the subunits that carry the activity of the proteasome, the beta subunits, I forgot to mention that although there are seven different betas, only three of them do carry an N-terminal threonine
and are active with somewhat different specificities. I forgot to say that. Now, these three or two times three active beta subunits in the immune proteasome are exchanged for immune subunits. All others are identical and also the exchange subunits are very, very similar,
but they have a few amino acid residue exchanges around the active site determining the specificity. So the immunoproteasome which is made in hematopoietic cells upon an immune stimulus inflammatory condition
and then the idea was then we could avoid or reduce the toxicity by targeting the immunoproteasome and open a new strategy against autoimmune disorders. So this is what we and others are now working on,
which is, well, autoimmune diseases are a terrible threat and there is no cure and the hope is that with inhibiting the immune proteasome, we open a new way, a new strategy for treatment.
Do you have time? We're running out. Yeah, just a few more minutes. Well, I just want to say this was all academic work and of course it's far outside of what an academic laboratory can do concerning drug development. So we looked for help by companies
and we found help. This is a spinoff of my department 20 years ago that has grown into a service company. This is what they do
and this is what they have available for customers and this is where they helped us. They do have on this list of what they call gallery structures where all the steps, protein production,
crystallization structure analysis is available and you can get an answer in just a few weeks. One of the targets they have available is the proteasomes and we collaborate with them. We also collaborate with a spinoff of the Max Planck Society, which is called Lead Discovery Center.
They have been established in order to help the academic groups in the Max Planck Society to develop ideas to a higher state so that they become interesting for big pharma. So we collaborate with them and they were quite successful.
They were quite successful. We were quite successful in making highly specific immunoproteasome inhibitors. Now this is one of the animal studies that they did showing that a xenograft of lymphoplastic cells
on a mouse then can reduce the tumor volume to zero with immunoproteasome selective inhibitors while potassium is completely inactive.
So very promising, but also in autoimmune disease like rheumatoid arthritis turns out that immunoproteasome inhibition offers a new way also in this disastrous disease.
This is the end. This is my hometown, Munich, not far from Lindau, the capital of Bavaria. You have here that Lindau is Bavarian. This is in the springtime.
You see the mountains, northern slopes, still snow covered. And I show this slide because the middle portion of it shows the old university, the Ludwig Maximilians University, where Roentgen and Laue, Laue is the discovery of
X-ray diffraction by crystals, the method which meant the foundation of structural biology and is the basis of all the work that I have told you 100 years ago.
Long time. Thank you. We have time for a few more questions. Are there any questions here? Oh, yeah. Thank you. Okay, so most of the inhibitors that are currently available
target the catalytic subunits. Have you ever tried to design an inhibitor or an activator that targets the gates rather than the actual catalytic subunits?
Well, this is our dream. We would like to have that. Definitely. It would be wonderful. And I think it would open new avenues for neurodegenerative diseases. I don't know, but we have now. And I don't know.
You see, you would have to open these entry gates. We know we can do it by making a mutation. I showed the picture, but you would need a small molecule that does do similar things. We haven't found anything. No, the inhibitors certainly don't do that.
They inhibit, but I'm not sure. I mean, there is a worldwide activity in inhibitor search for the proteasome, and I'm not sure whether the researchers there did look at activation, you see. They looked at inhibition and perhaps just overlooked activation.
Thank you for the talk. My question is regarding the immunoproteasome as a target in autoimmune diseases. And I raise this question because in cancer cells, regarding bortezomib and carfilzomib, resistance occurs at some point.
And they speculate also some reports that it is due to the other subcatalytically active subunits taking a part of the activity of the inhibited subunit. So in autoimmune diseases, this is an even longer therapy. So could it be that it's maybe not the best approach
because resistance or other proteases take over the activity of the proteasomes? Well, the few experiments that have been done together with this lead discovery center that I mentioned, of course these are short-term experiments,
and they are extremely promising, showing inhibitory activity where bortezomib is totally inactive. And you see a problem with bortezomib, it's inactive against solid tumors. One would like to have a proteasome inhibitor that is active against solid tumors.
So there is a chance with the class of compounds that have been developed for the immunoproteasome. So it's a very active field, and I just can say again,
a wonderful thing for young people to work on that. Yeah, and if I may, just one more question. So there are three catalytical subunits. The beta-5 is really well-defined, I would say, and we know how many roles it plays. But the beta-1i and beta-2i are really neglected. Do you have maybe any ideas where this...
Because we don't know much about it. Well, the beta-1 in the constitutive proteasome cleaves after acidic residues, and that is one of those changes in the immunoproteasome. There is an arginine in the constitutive that is responsible
for the acidic activity replaced by a leucine, so making it a chymotryptic specificity. That is the most prominent change in the immunoproteasome. So, of course, it would be most or almost all of the inhibitors
that have been developed target beta-5 or all of them, you see. We would like to specifically target beta-1 or beta-2. We may have surprises, perhaps,
a very different disease spectrum that we can address. So, it's a wonderful field of research. There's a question here.
So, it might be a silly question, but as far as everyone here knows, you are awarded a Nobel Prize for the discovery of the 3D structure of a reaction center, and now you're talking about a proteasome. So, how did you come to pursue another important project after your huge success in reaction center?
I think I have more other important projects, but I began my studies in protein crystallography, which was in the late 60s, early 70s, with basic pancreatic trypsin inhibitor called BPTI.
And then I stayed and my group stayed in the protease field since then until my emeritus group, which I have now. So, we always worked on proteases and had some side activities, and one of them was very successful.
That was in the photosynthesis. Thank you. But it was actually a side thing. Thank you. So, I have a very general question. So, given the rapid advancements in cryo-electron microscopy,
what do you think the future of x-ray crystallography is going to be in future research on the proteasome? Well, what is happening in cryo-electron microscopy is a revolution. It's now a novel, a new tool that we have. But it won't replace x-ray crystallography.
You see, the major drug targets, I showed you the list that is on this Proteros gallery structure. That list was made on what the customers want.
They do what the customers want, what they can sell. And these are 150 kinases and perhaps 100 proteases. And these are all relatively small molecules, so below 100,000. And this is where electron microscopy has problems.
They can do the very large objects. There's an easy explanation for that. But they have difficulties with the small ones. This is one point, and the other point is the resolution they have. You see, we have to inform, to give guidance
in the drug design process, guidance to the chemist. And the chemist needs, of course, a precise structure, precise interactions, so that they can then modify a hit
and develop into a lead. So in the near future, I don't see that electron microscopy. I admire it. It's wonderful to have it. But there is plenty of room for x-ray crystallography.
If people say cryo-electron microscopy is the end of x-ray crystallography, this is nonsense. Fantastic. Thank you all for coming and sharing this time together.