The Ubiquitin Proteasome Pathway

Transcription

1 [0:00:00] Slide 1 The Ubiquitin Proteasome Pathway Hello and welcome to today s Science/AAAS live webinar entitled, The Ubiquitin Proteasome Pathway: Targets for Disease Treatment and New Tools for Discovery. My name is Sean Sanders and I m the commercial editor at Science Magazine. Protein modification by ubiquitin or the ubiquitin like proteins has been implicated in a variety of cellular processes including proteasomal degradation, cell signaling, gene transcription, DNA repair, and protein localization. The nature, location, and extent of these posttranslational modifications appear to influence the cellular fate of the modified molecules. Recent evidence that small molecule inhibitors of the proteasome can be used in the treatment of human cancers has launched intensive efforts to discover new drugs that will selectively inhibit the activities of disease specific components of the ubiquitin pathway. It has also stimulated the development of new tools to help better understand the complex mechanisms surrounding ubiquitin and the ubiquitinproteasome pathway. In today s educational webinar, we will be learning more about the ubiquitin proteasome pathway including the importance of ubiquitin ligases in the signaling process and their roles as targets for drug therapy. We will also investigate some of the current technologies for studying this important cellular pathway. In the studio with us today, we have three eminent experts in the field. Sitting just to my right is Dr. Alfred Goldberg from the Department of Cell Biology at Harvard Medical School in Boston. Next is Dr. Gregory Korbel from Invitrogen s Bradford, Connecticut site. Last but certainly not least, we have Dr. Paul Andrews from the Cambridge, Massachusetts Campus of Amgen. Before we kick off with our first presenter, I wanted to remind all of our viewers that if you wish to see a more enhanced view of any of the slides presented today, you can click on the enlarge slides button located just underneath the slide window of your web console. If you like, you can also download a PDF copy of all the slides using the download slides button. To submit a question live to the panel or to a particular speaker, simply type it into the ask a question box at the bottom left of your viewing console and then click the submit button. We ll do our best to 1

2 get to as many of the questions as we can. Keeping them short and to the point will give you the best chance of having them answered. We d like to thank Invitrogen who is kindly sponsoring today s webinar. Now, on to our first speaker. Dr. Alfred Goldberg received his A.B. degree in biochemistry and his Ph.D. in physiology from Harvard University after attending Harvard Medical School and Cambridge University as a Churchill Scholar. He has been on the faculty of Harvard Medical School for nearly his entire academic career, and since 1993 has held the position of Professor of Cell Biology. His laboratory first demonstrated the nonlysosomal ATP dependent pathway for protein breakdown, now termed the ubiquitin proteasome pathway. Dr. Goldberg and his colleagues were the first to introduce proteasome inhibitors now widely used as research tools. His work led to the development of the drug, Velcade, now widely used in the treatment of multiple myeloma. Dr. Goldberg s accomplishments have been recognized with many awards and he has served on the scientific advisory boards of a number of pharmaceutical and biotechnology companies. Dr. Goldberg? Dr. Alfred Goldberg: Thank you, Sean. Slide 2 Today, we re going to be discussing the possibilities of drug development in the ubiquitin proteasome pathway. Perhaps one of the major conceptual advances in cell biology in the last 20 years has been the recognition of the importance of protein breakdown in controlling cell growth, in controlling cellular responses, and quality control. And this kind of selective breakdown of certain cell proteins is made possible by the pathway for protein breakdown now termed the ubiquitinproteasome pathway. About 30 years ago, it became clear that protein breakdown was not just occurring within the lysosome, which mainly degrades extracellular endocytosed proteins. Intracellular proteins in the nucleus, in the cytosol are continually being degraded by an ATP dependent pathway. And attempts to understand why there is this requirement for ATP led to the discovery of the involvement of ubiquitin. There are two main phases for the ubiquitin proteasome pathway. The work by Hershko and Ciechanover for which they won the Nobel Prize in 2004 indicated that proteins targeted for degradation are modified 2

3 covalently by linking a chain of ubiquitins. This is a multi enzyme ATP acquiring process about which you ll be hearing a great deal. Slide 3 [0:05:27] Slide 4 In addition, there is another energy requiring step involving a huge intracellular complex called the 26S proteasome. It binds the ubiquitin chains, unfolds the protein, and degrades them to short, short pieces that emerge from the proteasome, but last only seconds because they rapidly hydrolyze in the cytosol to free amino acids. Some of these are also used in the process of antigen presentation by which the immune system screens for viruses in cancer. Now, the ubiquination process is summarized in the next slide. It is a three well basically three main steps, the first of which is an energydependent activation of the ubiquitin carboxyl group to make it highly reactive, to make it a thioester intermediate. This step leads to the transfer of the reactive ubiquitin to one of the cell s 25 to 35 different ubiquitin conjugating enzymes or E2s. They serve to transfer the ubiquitin to the substrate in collaboration with one of the ubiquitin ligases. Here is where the real exquisite selectivity of the process emerges. We now know that humans contain at least 650 perhaps a thousand different ubiquitin ligases, far more than the number of kinases in our genome. These enzymes bind the substrate, specific substrates or a group of substrates, and link them to a chain of ubiquitin molecules very rapidly, which leads to their ultimate degradation by the 26S proteasome. Slide 5 But the next slide illustrates what kind of selectivity one has in cells, and these are some of the most interesting proteins in cell biology. These are the short lived components in cells and many of them represent very attractive targets for development. Oncogene products or the tumor suppressors such as p53 or Mdm2, major targets of medical interest, are rapidly degraded with half lives as short sometimes as 15 to 20 minutes. Or it can be taken as a generalization that almost all important regulatory proteins have short half lives. Cell cycle is regulated by the program degradation of inhibitors of specific cells, cellular transitions between stages in the cell cycle, and also by cyclins degradation. 3

4 Almost all transcriptional regulators have very short lived proteins. The most interesting of which is NF κb or its inhibitor, IκB. HIF, which controls the response to ischemia. β catenins, which control many cancer responses. All are very short lived proteins, which mean they have specific ubiquitin ligases involved in their degradation. This extends to many other areas of biology. For example, our lab is very interested in the muscle wasting that you see in cancer patients, in bedridden patients or if you have an arm in a cast. Slide 6 And the next slide shows work from our lab and from David Glass group that there are two main ubiquitin ligases that are absolutely essential for rapid atrophy of muscle. This slide is to illustrate that there are tissues specific to ubiquitin ligases and these enzymes can be dramatically induced in disease states. It is also meant to illustrate that ubiquitin ligases, as in the left hand case of MURF, can be a single polypeptide that binds the substrate and binds the ubiquitin E2 pair. Other ubiquitin ligases such as the SCF complex are multi subunit modular complexes, and we still don t understand what advantages this complex architecture provides. Slide 7 Slide 8 Slide 9 Slide 10 The proteasome is an intriguing, we think, beautiful structure shown on the next slide. This is an EM tomograph from the work of Baumeister of the human proteasome, which is really identical to the frog or the butterfly proteasome. It s composed of 65 or so individual subunits. The core proteasome, this cylindrical shaped yellow image, is where proteins are degraded to small pieces. And you can see the core outside is a tightly inter woven subunits such that proteins and peptides cannot transit except through small openings at either end. The 19S regulatory component binds the ubiquitin chain as on the next slide, and this still a poorly understood process. There are 6 different ATPases whose function, we believe, is to unfold the protein and to translocate, it like a syringe injected into the central core proteasome where the proteins are degraded to small peptides. And as we mentioned, they are quickly converted to amino acids. The main point is that the protein is digested, the ubiquitin chain is disassembled by the 4

5 deubiquitinating enzymes that you ll be hearing about, and the ubiquitin moieties are recycled for further rounds of degradation. [0:10:35] Slide 11 The proteasome offers many possibilities for drug development, but so far, these efforts have focused on the proteolytic core. The next slide shows a cross section in the bottom phase of the 26S proteasome. You can see the central 20S particle, the cylindrical core, and it has central chamber inside of which proteins are digested. We call that the center of doom for the protein because it has the proteolytic sites. At either end in the 19S regulatory core, there are two orange rings, which are the ATPases. It was shown above that the proteasome complexes from the earliest organisms, archaebacteria, which have a very similar architecture to the proteasomes in our cells so this has been a highly conserved mechanism. And there are very similar ATPase rings, which would have been advantageous for studying mechanisms. The difference in eukaryotes is the lid, the white at either end, which has the machinery for binding the ubiquitin chain and for disassembling it. In eukaryotes, protein breakdown depends on ubiquination, which provides all the selectivity of the process. In prokaryotes, some of which have proteasomes, this process is independent of ubiquitin, proteasomes in existent organisms such as tuberculosis, and they may also be future targets for selective antibiotic therapy. We re very interested in the 19S regulatory complex because we think it offers additional targets for drug development. The binding of individual ubiquitin chains, the unfolding process, and also the opening of a gate. Slide 12 It turns out as shown in the next slide, which on the left is an x ray diffraction image that shows the either end, the outer alpha ring of the proteasome. And you can see the central hole through which proteins have to be translocated to be degraded. They are injected by the ATPases through that central pore. On the right shows some recent work from David Smith and Yifan Cheng in our group that actually shows how the gate exists normally in a closed form that keeps proteins from the cytosol out of the proteasome, unless they are delivered correctly by the 19S. 5

6 Slide 13 Slide 14 The 19S complex opens the gate as shown in the next slide, which allows the protein to enter, and this is an ATP dependent reaction. So, if the ATPases close the gate and they hydrolyze the ATP to ATP, and they open it when another appropriate substrate comes along. We re very interested in this and target this process as a potential target for drug development. But the main point I just should end with is to tell you about these new agents for the treatment of hematological cancers that affect the proteasome s active sites. In mammalian or other eukaryote proteasomes, the 20S has in its central chamber of doom six active sites. In each ring, each of the β rings, there is a chymotrypsin like site that cleaves after hydrophobic bonds, a trypsinlike site that cleaves after basic groups, and a caspase like site that cleaves after acidic amino acids. So, the proteasome like the lysosome of the pancreas has the ability to cleave many different kinds of peptide bonds. When we were developing the initial proteasome inhibitors, we didn t know any of the structural information that I ve just told you. But we did know that the chymotrypsin like site was a particularly important one and we knew that small hydrophobic peptides could get across cell membranes. So, the first drugs were simple homologues of this substrate shown here, the Leu Leu Val Tyr substrate commonly used to assay the proteasome. Slide 15 [0:15:25] The next slide illustrates some of the first proteasome inhibitors that emerged. MG 132 has now been used in over 3000 scientific papers because cause it s an easily reversible blocker of protein breakdown. And the structure may look formidable, but it s only three leucines in a row with a phenoxy group to help inactivate the amino terminus to help it get across membranes and an aldehyde, the warhead that inhibits the active site of the proteasome. Through the innovation of Julian Adams who led the medicinal effort chemistry effort, that aldehyde was replaced by a boronic acid, which allowed a tremendous increase, maybe a hundredfold increase, in efficacy. And that led to the ability to inhibit the proteasome but not other proteases in our body. The proteasome has a unique mechanism. It has a threonine in its active site nucleophile. 6

7 Also shown here is a vinyl sulfone derivative. That s a covalent inhibitor of the proteasome developed by Matthew Bogyo and Hidde Ploegh. And on the bottom lactacystin, which is an antibiotic produced by actinomyces and a derivative of lactacystin. A natural product also is going to be going into human patients or maybe already in phase 1 trials. Slide 16 What I should just point out is that MG262, the boronic acid, was the parent for Velcade. The medicinal chemists were able to reduce the size and generate this molecule now called bortezomib by its generic name or PS 341, which was the original name from, I guess, that company s name was ProScript, PS. Now this is a remarkably effective new treatment for multiple myeloma. It would soon be a billion dollar drug because it has greatly given hope to patients with this and certain related cancers. It has a number of actions that make it attractive as an anticancer agent. The most important of which for the hematological malignancies is that it inhibits it prevents the activation of NF κb. NF κb is essential for the transcription of genes for IL 6, the growth factor for these cells as well as certain factors that the myeloma cells need to develop and to attach to bone marrow. Also, the myeloma cell is a cancer of the plasma cells. It s producing abnormal immunoglobulins. And the ubiquitin proteasome pathway has special quality control mechanisms to degrade rapidly misfolded secretory proteins such as misfolded immunoglobulins. This is very important in normal plasma cells. It s particularly important in these cancers. When this process is blocked by bortezomib, then these misfolded proteins accumulate and they activate JnK kinase in apoptosis. So, the lack of NF κb, which inhibits apoptosis, and this triggering of cell death lead to a special sensitivity of these hematological malignancies. Slide 17 This agent was sufficiently effective that it was approved by the FDA after only phase II trials. Now, it s been approved as a third line therapy I mean second line therapy. And it s very likely in the next few months, it ll be approved as a first line therapy in combination with other agents. Because recent trials have shown that over 90% of the patients seems to be benefitting. Two other rather rare lymphomas, mantle cell lymphoma and Waldenström macroglobulinemia also respond very strongly. And there 7

8 are presently at least 200 trials ongoing for solid and other hematological malignancies using this agent. Slide 18 Slide 19 [0:20:03] Other companies are pursuing different proteasome inhibitors, all of which affect the same active site, the chymotrypsin like site within the 20S proteasome. And they are now also very promising agents in clinical trials. What I should end with is one last point. Proteasomes serve in addition to this role in cell regulation; a special role in immune surveillance. As I mentioned, oops let s skip that slide, just go on to the immune surveillance one. What we show here is that there are two arms of the immune system. The immune system actually respond not to proteins but to fragments of proteins, peptides, extracellular protein antigens degraded by the lysosome to peptides, and that elicits immune responses, antibody production through MHC class II molecules. But the cell also is virtually all cells of our body are continually screening for abnormalities in intracellular space; the appearance of viruses, intracellular pathogens, cancers. And the way that works, as we indicated, is fragments emerging from the proteasome are transported into the endoplasmic reticulum, trimmed down to small peptides that are eight or nine residues long, and are bound to MHC class I molecules that goes through the cell surface. If a foreign peptide is presented then the cytotoxic T cells will destroy this cell. So the garbage collecting mechanisms of the cells have been utilized as an informational system. But also, the point I want to end with is that the proteasomes in these cells tend to be specialized. There is an immunoproteasome that s activated in inflammatory states and is specific for immune cells. There are special activators of the proteasome found in these cells. And so a very attractive drug target in the future for the treatment of immune disorders and inflammatory disorders are selective inhibitors of the immunoproteasome mechanism. And at that talk, I guess, the main take home lesson I m trying to leave you with is there are many opportunities emerging here for therapeutic interventions based on knowledge of this fundamental cellular pathway. Thanks. 8

9 Thank you very much, Dr. Goldberg. So, we ve had some questions come in already during your talk and I m going to pose a couple of them to you right now. How quickly or slowly does protein degradation following ubiquitination occur, and are large proteins degraded more slowly than small proteins? Dr. Alfred Goldberg: Yes. The screening step, the role of the E3s in binding the substrate is the rate limiting step in the process. Once one attaches a ubiquitin chain, the proteins typically only have seconds before they bind to a proteasome and degrade it under normal conditions. The proteasome itself also degrades proteins, even long ones, within time shorter than a minute. That process is a bit longer for long proteins than it is for short proteins, but the E3s are really the rate limiting step. Okay. Excellent. And a follow up question to that is are ubiquitinated proteins able to function following ubiquitination, and are they always degraded if they ve ubiquitinated? Dr. Alfred Goldberg: Yeah. That s a very important question. If proteins were automatically inactivated when the proteasome was nonfunctional by ubiquination, then these drugs would be very toxic to every cell in the body. Uh huh. Dr. Alfred Goldberg: When the proteasome activity is reduced, proteins continue to be reubiquinated. But that s another process that the subsequent speakers will be talking about in which such proteins can be deubiquinated and are functional again. And as far as we know it, ubiquitin has a tag that doesn t even affect function. You can show enzymatic activity maintained in these few cases where it s been looked. It s a reversible phenomenon. Slide 20 Great. Okay, we re going to move quickly on to our second speaker today and this is going to be Dr. Gregory Korbel. Dr Korbel completed his Ph.D. in organic chemistry in the Department of Chemistry and Chemical Biology at Harvard University. His postdoctoral training was carried out at Harvard Medical School and the Whitehead Institute for Biomedical Research at MIT, and focused on deubiquitinating 9

10 enzymes and pathways for ubiquitin mediated and non ubiquitinmediated degradation of misfolded proteins in the endoplasmic reticulum. He also trained at the Yale School of Medicine, characterizing the role of SUMO and SUMO proteases in cellular senescence. Dr Korbel joined Invitrogen in June 2007 and is currently Senior Scientist and head of ProtoArray Services. Dr. Korbel, welcome. Dr. Gregory Korbel: Slide 21 Slide 22 Thank you very much, Sean. So, I d like to dive right in to a little bit more of an overview of the ubiquitin proteasome pathway. Now, ubiquitin is an essential and highly conserved 76 amino acid protein, which can be appended posttranslationally to target proteins. This process is mediated by an enzymatic cascade, as Dr. Goldberg pointed out, involving an ATP dependent E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzymes, and E3 ubiquitin ligases. [0:24:52] Now, proteins that are tagged with ubiquitin, those tags can be removed. This process is reversible. And that is an enzymatic reaction, which is mediated by a family of proteins collectively referred to as deubiquinating enzymes or DUBs. Modification of ubiquitin on substrate proteins can occur in a variety of flavors. One of these is the attachment of a single ubiquitin unit and this is a process referred to as monoubiquitination. Monoubiquitination has effects on cellular localization as well as enzymatic function. The other main flavor of ubiquitination is a process referred to as polyubiquitination in which multiple ubiquitins are attached in a string on a particular substrate. A ubiquitin has seven internal lysines, each of which can be used to generate this polyubiquitin chains. And depending on the lysine of ubiquitin, which is used in this process, very different cellular outcomes can result. For example, if ubiquitin chains are constructed using lysine 63 of ubiquitin, these signal for intracellular trafficking events of membrane proteins, transcriptional activation, DNA repair, and some enzymatic activation processes as well. 10

11 In contrast, when proteins are modified with polyubiquitin chains that are linked through lysine 48 of ubiquitin, these serve as signals for translocation to and degradation by the 26S proteasome. As Dr. Goldberg mentioned, these proteins are then degraded into oligopeptides. Now there s an important facet here, which I ll point out now, and that is that the proteasome has associated deubiquitinating enzymes. And the function of these enzymes is to remove ubiquitin from substrate proteins so that ubiquitin can be very efficiently recycled into the ubiquitination cascade pathway. The ubiquitin proteasome pathway is essential to many, many aspects, nearly all aspects of cell biology. And as a result, particular components of these pathways are very important as attractive drug targets, and these include E3 ubiquitin ligases, deubiquitinating enzymes as well as the proteasome. Slide 23 One of the challenges, which is really yet to be overcome in this field is selectivity in downstream effects in targeting particular components of the pathway. For example, to date, clearly the most successful clinical target has been the proteasome. Indeed, Velcade has truly revolutionized the treatment of multiple myeloma. However, the proteasome is responsible for degrading a very large number of proteins and therefore targeting this particular protease, in theory, affords relatively little selectivity in the inhibition of downstream effects. So, in contrast to the single proteasome, there are at least two ubiquitin E1 activating enzymes, which activate ubiquitin and transfer ubiquitin to dozens of E2s. These E2 conjugating enzymes then act in concert with E3 ubiquitin ligases, many, many more E3 ubiquitin ligases to target substrates for ubiquitination. Due to the hierarchical design of this pathway, it implicates E3s as really the point of maximal selectivity in targeting this pathway effectively. E3s have been implicated in a number of disease, cancer and immunological disorders, a large and growing number of conditions. However, the substrate specificity of E3s really remains largely unknown. The important point though is that substrate identification of E3s is critical for linking these E3s to specific biological pathways. Slide 24 So shown on this slide is an overview of the main strategies that are used to identify ubiquitin ligase substrates. Many of these same strategies are also used to determine substrates for deubiquitinating enzymes. It s 11

12 really techniques that are relevant to the pathway as a whole. Unfortunately, there s relatively a limited number of strategies that have been described. But these include for example endogenous gene mutations and deletions, which help to establish links between gene alteration and pathology. Also relevant to these are viruses. Viruses have been instrumental in helping us to elucidate parts of this pathway. There s also the aspect of changing E3 ligase expression levels either through overexpression or RNAi based knockdown strategies. And these can influence cell behavior and function or morphology. They can also change the ubiquitination or degradation profile of the proteome in those cells. [0:30:12] Now a lot of work has been done with proteomics approaches and these really involved a variety of strategies, contributing strategies, such as coimmunoprecipitation, 2D gel electrophoresis, quantitative mass spectrometry. And this has really been important in helping to identify proteins that bind the ubiquitin ligases as well as to co purify with them as larger complexes. It s also been very useful in identifying the ubiquitination and degradation profiles as well as in evaluating the particular linkages that are used to construct polyubiquitin chains. There are also some biochemical assays, in vitro assays, which make use of purified proteins and protein fractions in conjunction with labeled ubiquitin derivatives. And in this manner, you can follow the modification of substrates with ubiquitin using spectrometric techniques such as luminescence or FRET (Fluorescence Resonance Energy Transfer) or even by standard Western blot analysis. A more recent addition to the toolbox here is protein microarrays. In this case, these can be quantitated simply by using a standard fluorescence detection system of a microarray scanner such as you might use for DNA microarrays. Slide 25 Now, Invitrogen has a functional human protein microarray, which contains over 8000 human proteins. These proteins are expressed as GST fusions in insect cells, and they are part of the Ultimate ORF collection of Invitrogen where all of these clones have fully sequences and validated. These proteins once expressed are purified using the GST tag and are then printed on to nitrocellulose coated glass slides. 12

13 The content of these human protein microarrays is illustrated in the text box on the bottom left. And this is meant to provide an overview of the types of functional classifications of proteins that are on the array. And there really is a wide range of proteins that cover a very wide range of biological processes. Slide 26 Now, shown here is an expanded view of what the protein microarray looks like after probing with an anti GST antibody and a secondary detection reagent. The array is composed of 48 individual subarrays in a 20 x 20 arrangement where each protein that has been purified is deposited on the slide as adjacent duplicate spots. In addition to the human protein content, there are a number of control spots, which are used for quality control purposes as well as for application specific positive and negative controls. Slide 27 Slide 28 So now, I d like to describe some of the strategies using human protein microarrays in the identification of ubiquitination targets. The assay itself is a very simple and rapid assay. It involves blocking of the nitrocellulose membrane with a blocking agent, very similar to the way that you would treat for Western blot analysis. And in this manner, you prevent nonspecific adsorption of components of your biochemical reaction of interest. The array is then incubated in a solution of biotinylated ubiquitin in conjunction with E1 enzymes, E2 enzymes, and E3 ligases. Now following the ubiquitination reaction, these arrays are washed and then probed with a secondary detection reagent specific for the biotin, in this case, a fluorophore labeled streptavidin. The arrays are then washed and dried and scanned, much as you would do for DNA arrays. So, in addition to the operation simplicity of this particular process, there are other benefits as well. For example, each array requires a very small quantity of E1, E2, and E3 enzymes, and this really helps to conserve on precious protein materials so that they can also be used in a number of other assay formats. In addition, the assay is robust and you can use enriched fractions for particular types of proteins or you can use purified components as well. And another big advantage here is the time to completion. The assay 13

14 takes only four hours to complete from start to finish. So in one day s time you really can start your assay and get to the point where you re analyzing and looking for new targets of ubiquitin ligases. [0:34:57] Slide 29 So shown here is an example of a human protein array, which has been probed with rabbit fraction II. This is a cell lysate fraction, which is enriched for ubiquitination machinery. And this was incubated with rabbit fraction II and biotinylated ubiquitin and subsequently, the detection, as I mentioned previously, was with a fluorophore labeled streptavidin derivative. Now in this particular experiment, a number of ubiquitinated proteins were observed. These included a wide range of functional classifications including the three, which as shown here, which are E3 ubiquitin ligases, known E3 ubiquitin ligase substrates as well as novel substrates of E3 ubiquitin ligases. Now shown on the bottom left panel are the three examples. There s E3A, which is a known E3 ubiquitin ligase. And this is something for which the protein is probably auto ubiquitinating itself on the surface of the array. And it s actually a very nice demonstration of functionality of the proteins when they re deposited on the surface of the slide. Rad23A is a protein, which is involved in DNA repair, and this is known to be ubiquitinated in vivo. And so this is a nice validation of finding known targets and it suggests that the proteins that are on the array are actually folded and functional. Finally, HGS is a protein, which is in involved in scaffolding complexes for receptor complex and signal transduction. And this is actually a substrate, which has not been previously reported to be ubiquitinated suggesting that it s actually a novel E3 ubiquitin ligase substrate. Now shown on the bottom right is some of the data, which has been presented in graphical format. And this shows that you don t observe ubiquitination unless all of the necessary components; the E1, the E2, E3, and ATP are present. Slide 30 This is an example of profiling yeast protein arrays. In this case, this was work, which was done at the University of Toronto, and this work evaluated the yeast ubiquitin ligase, Rsp5. In this experiment, the arrays were probed with E1, E2, and E3 enzymes in the presence of a fluorescein conjugated ubiquitin derivative and a number of substrates were 14

15 identified. In fact, 150 candidate Rsp5 substrates were identified, a subset of which is shown on the bottom left panel. Some of these were followed up for in vitro ubiquitination assays and it s very important that all them were validated in these biochemical assays. Indeed, three substrates, which were investigated for in vivo effects were also validated by that experimentation as well. Slide 31 Dr. Gregory Korbel: So now I d like to move on to a related process and Excuse me. Dr. Korbel, I m going to have to interrupt you, we re running short on time so Okay. maybe we can skip over these Dr. Gregory Korbel: Sure. the SUMO slides and Dr. Gregory Korbel: Dr. Gregory Korbel: Slide 34 Dr. Gregory Korbel: Dr. Gregory Korbel: Sure. Okay. you can go on to your summary? Absolutely. That d be great. Thank you. So it really is important that I hope we ve been able to identify some of the very exciting aspects of ubiquitin and ubiquitin like biology. Because these are very important pathways to the cell and provide new insights not only to cell biology, but also can help broaden our understanding of many diseases as well as provide potential new drug targets, which Dr. Andrews will talk about shortly. Great. Thank you so much. Thank you. Sorry for interrupting you. We want to try and get to our Q&A session, which I m sure everyone is very interested in as well. 15

16 Dr. Gregory Korbel: Slide 35 Uh hum. So, we re going to move on to our next speaker. Our final speaker today is Dr. Paul Andrews. Dr. Andrews received his Ph.D. at the University of North Carolina at Chapel Hill where he studied the substrate targeting mechanisms of the RING finger and cullin based family of E3 ubiquitin ligases. He is currently a scientist with the Lead Discovery organization at Amgen where he has expanded his exploration of the ubiquitinproteasome pathway. Dr. Andrews? Slide 36 Dr. Paul Andrews: Slide 37 Thank you, Sean. So, I m going to introduce to everybody some of the novel technologies that are being used for the discovery of new therapeutics targeting the ubiquitin proteasome pathway. Now I m not going to through the pathway once again, as our other panelists have already done so. But what I would like to do is kind of reiterate the potential points of intervention for small molecules in the ubiquitin proteasome pathway. And you can imagine that you could target the enzymatic activity of the E1, the activity of the E2, the E2 E3 interaction, the catalytic activity of the E3 or the E3 substrate interaction. Now you can also target the activity of the removal of ubiquitin by the deubiquitinating enzymes, and of course, you can target the 26S proteasome. And this is what our previous panelists had talked about in Velcade. And Velcade to date is still the only FDA approved, therapeutic for intervention within this pathway. [0:40:12] Now we at Amgen are particularly interested in looking at inhibiting the activity of the E3 ligase because this is where the selectivity within the pathway lies. Slide 38 So here, I ve listed a number of technologies that are available for the identification of small molecule inhibitors of the enzyme cascade in the ubiquitin proteasome pathway. And this is by no means all inclusive. These are just some of the technologies that we have thought about utilizing. 16

17 So, for E1s, E2s, and E3s, there are a number of ATP loss or ATP detection technologies, and these are really suited nicely for identifying inhibitors of the E1. There are also a number of TR FRET based technologies. These are technologies such as CisBio s HTRF, Invitrogen s Lanthascreen, and Perkin Elmer s AlphaScreen. And Perkin Elmer s AlphaScreen isn t really a FRET based technology, but it s very similar in the sense that upon excitation of the donor bead, it releases a single oxygen that is then transferred to an acceptor bead if it s within close proximity. So, it operates in the same principle as a FRET based assay so I ve grouped those together. And then there s electrochemiluminescence or ECL, which is produced by Meso Scale Discovery. And really the TR FRET and the ECL based technologies are really suitable for identifying E2 and E3 inhibitors. So, there are also a number of technologies available for identifying deubiquitination inhibitors, kind of the classic reagent is ubiquitin AMC. And then there are a couple of novel technologies such as Progenra s ubiquitin ISOpro technology and Invitrogen s Lanthascreen. And I ll show some data on the Progenra technology later on in my talk. So many of these technologies can be utilized for the interrogation of ubiquitin pathway, but also for the ubiquitin like protein molecular pathways as well such as SUMO and NEDD8. Slide 39 Slide 40 So here, I ve listed three technologies that we ve looked at for identification of ligase autoubiquitination inhibitors. And these are the Perkin Elmer AlphaScreen, the MSD Electrochemiluminescence or ECL, and the Invitrogen Lanthascreen. I m not going to discuss any one of these technologies in detail. If anybody wants to know more information on any of these technologies, they can go to the respective websites of these companies. So, as I mentioned, we re interested in identifying small molecule inhibitors of E3 ligase autoubiquitination. So, this is a representative subset of compound data from one of our screening campaigns where we compared the MSD ECL technology and the AlphaScreen technology. And as you can see we took a this is just a small subset, compounds A, B, and C. We put these through each one of the technologies and at the top, I ve put there the IC50 graphs for each one of these compounds A, B, and C. And both technologies revealed similar IC50 values for the majority of the compounds tested, not just these three compounds here. And you 17

18 can see that in my table on the bottom left, I ve listed these IC50 values for compounds A, B, and C in the MSD and AlphaScreen technologies respectively. Now these technologies can both be used for studying the ubiquitination of substrates as well if the substrates are known. But for many ligases that sit on molecular pathways involved in disease states, the substrates for these ligase are not known. Slide 41 So, what we set out to do is to utilize protein arrays to identify these substrates of the potential ligases with the substrates that are unknown. Now this is a proof of principles experiment that we did with Invitrogen s ProtoArray. And what we did here was we took an E3 ligase that we knew had two substrates that were very well characterized. We printed these out on the arrays, and then we performed ubiquitination assays to see if we could ubiquitinate these two known substrates. We also utilized titration of E2 to try and identify the optimal E2 and E3 substrate complex within this experiment. And as you can see in columns 1, 2, and 3, there is no ubiquitination of substrate 1 or substrate 2 without the addition of E3. Now, there is ubiquitination of the E3 and this nice because this takes advantage of certain some E3s have the ability to autoubiquitinate. So this is a nice internal control because this controls for all the components of the assay and it demonstrates that all the protein components work. [0:45:23] Slide 42 So, I ve also outlined a number of other controls that are printed on these arrays to control for various aspects of the detection, and those are outlined in red. And as you can see in column 4, both substrates were ubiquitinated only with the addition of the E3. So, you can see here that substrate 1 and substrate 2 are ubiquitinated by the E3. And I ve represented this below as a graphical summation because probably the ProtoArray raw data itself is kind of light. But you can see in the graphical representation that substrate 1 and substrate 2 with increasing concentrations are ubiquitinated with the addition of the E3. In addition, the E3 itself has an increase in its ubiquitination with the increased concentration of E3. Now the ultimate goal of this study was to identify substrates of this particular E3. Slide 43 18

19 So, we then went ahead and probed the Invitrogen ProtoArray human microarray to look at if we could identify substrates of this particular E3. At the top in the box, I have a representative E3 substrate ubiquitination data set. As you can see, we have nice signal in the background that titrates in with addition of E3. Now, in this experiment, we looked at a couple of different E2s to see if we could identify selectivity with the E2 E3 substrate pairings or complexes. And we identified with E2 #1, 86 proteins that were ubiquitinated and with E2 #2, 33 proteins that were ubiquitinated. Now some of these did overlap and they weren t completely unique, but a majority of those proteins were unique and selective for the different E2s. The other nice thing about the array is because it contains so many proteins on the array, there were known E3 substrates that were on this ProtoArray that we did identify as substrates in our own study. So, our preliminary results indicate that the ProtoArray is a useful tool for identifying novel substrates utilizing recombinant mammalian components. But there s still a lot of work that we have to do to validate a lot of these substrates and those are experiments that are still ongoing. Slide 44 So finally, I d like to finish up showing you a representative data set of a screening campaign that we looked for small molecule inhibitors of deubiquitinating enzymes. Now, the technology that we used was Progenra s Ubiquitin ISOpro assay. And briefly, what this assay technology consists of is ubiquitin reporter fusion protein. When incubated with a deubiquitinating enzyme, the ubiquitin is cleaved releasing an active reporter, which can then go downstream and act on its substrate giving it a signal that we can quantitate. I have just a couple of compounds that I ve demonstrated their IC50 curves, and as you can see, the assay gives reasonable signal in the background. The only caveat to this assay is that it is a coupled assay so you do need to assay against a free secondary reporter to ensure that you re not inhibiting the secondary reporter and you are inhibiting the deubiquitinating enzyme. Slide 45 Finally, I d just like to acknowledge the scientists involved in these studies, Erin Mullady and Steve Schneider who performed some of the 19

20 work. And the scientists Renee Emkey, Dave Green, and Philip Tagari who enabled us to do this work. Dr. Paul Andrews: Slide 46 Great. Thank you, Dr. Andrews Thanks. Some very nice data. So, we re going to move on to our Q&A session now. As I ve said, we ve been running a little bit long so we re probably going to go a little bit over time. I hope the audience stays with us for this. I think the Q&A session is going to be very informative. So, I m going to jump right in with a question for you Dr. Andrews. Can these arrays that you ve used be used to investigate compounds that inhibit E3 ligase activity? Dr. Paul Andrews: [0:50:11] Actually, that s a great question. And the ultimate goal in our studies is to be able to use the arrays in such a manner that when you do identify selective or potentially selective inhibitors for a particular E3 ligase, that you can imagine you can take these arrays, either the full content protein arrays or custom arrays where you have known substrates. And you can go ahead and probe those arrays using the inhibitors to see if there s actually selectivity of the different inhibitors for inhibiting certain substrates. So, you can add another level of complexity in there swapping in and out E2s to see if you can selectively inhibit an E2 E3 substrate complex. But those are ultimately the goals for us to use that as a tool. Okay. Excellent. So, I m going to come back to one more specific question and then we re going to have a more general question that you can all discuss. This is for Dr. Korbel. Somebody is asking the evidence that the proteins on the ProtoArray are functional. Do you have some evidence to provide? Dr. Gregory Korbel: Uh hum. Actually, I think the best evidence comes directly from the assay where we see that proteins, for example E3 ubiquitin ligases, which are present on the array, will autoubiquitinate in the presence simply of E1, E2, and ATP. In addition, I think another good piece of evidence is that we identified in these assays proteins that are known to be ubiquitinated in 20

21 cells or even in animal models. It s really a nice validation to be able to find those proteins because it suggests that the proteins that are on the array are folded properly, can be recognized by E3s, and are functional when they re on the array. Okay. Good. So, a more general question that maybe I ll start with Dr. Goldberg. You can have a go at this one. Somebody was asking about the current state of the art for drug inhibition at the moment in this system. Dr. Alfred Goldberg: As the three of us have emphasized, at the moment, there is one agent out there that s proved very useful, (bortezomib) Velcade for hematological malignancies. What s been a surprise and a little disappointing is that it was very effective in animal models against solid tumors, but so far, that promise hasn t held up in human trials. But it may depend on the cocktail of inhibitors. There are many cases where an agent, for example, induces a cellular stress response where the proteasome plays a critical role. And mixing of multiple inhibitors has proven particularly promising in achieving cytotoxicity against cancers in vitro but not in vivo. The proteasome inhibitors also offer promise against many inflammatory diseases because of the key role of NF κb in those conditions in the induction of the immune response in proteasome function, and ubiquitin ligases are key there. I mentioned, there are three proteasome inhibitors in clinical trials aimed at the same diseases. The only other agent that is now close or about to enter human trials is from Millennium, and it inhibits the E1 not of ubiquitin, but of a ubiquitin like protein called neddylation. We haven t had time to mention that there are several ubiquitin like proteins in cells that undergo a similar dependent E1, E2, E3 dependent ligation. And an inhibitor of the neddylation actually affects the activity of a group of ubiquitin ligases. And this agent looks like an attractive approach for treatment of certain cancers. The rest of the field is basically at the pre clinical stage. I think, the takehome lesson of all three speakers is that there s an enormous number of targets, in cancer, in various metabolic diseases, in neurodegenerative diseases, which if you could inhibit or activate components of this pathway, you should have a therapeutic result. So now, the challenge is to find out more about these enzymes and find the molecules. 21

22 Dr. Gregory Korbel: Dr. Korbel, you d like to add something? I definitely agree with what Dr. Goldberg had mentioned. When it comes especially to ubiquitin like proteins, ubiquitin is one parent member of a much larger super family of proteins. But we really we don t know everything clearly about the ubiquitin proteasome pathway and we even know less about these ubiquitin like modifiers. But things for example such as SUMO or small ubiquitin like modifier, that has been demonstrated to have roles in cellular senescence. So that brings up the potential interplay with not just cancer, but also aging and aging responses. I think the door has just been opened and the best is certainly yet to come. [0:55:03] Dr. Paul Andrews: To add to that, to address kind of the state of the art, I think it s, as we all know, a very, very complex and still somewhat unknown mechanism for all of these pathways. I think that it s only recently that technology has been able to catch up to being able to assay and look at these specific very, very complex problems. And I think really that now has enabled us to move forward both in the academics and in industry to start answering some of these very difficult questions. Okay. So, here s a question that I think is interesting. We ll see what your opinion is. This viewer asked, is there any evidence for a relation between ubiquitination and phosphorylation? Dr. Alfred Goldberg: Yes, I think that s a very active area of research. I think it s fair to say that phosphorylation dephosphorylation is one of the major modes of cell regulation that s rapidly reversible. Ubiquination leads to the destruction of protein, which is for sometime irreversible until there s new gene expression. And now, there are many cases where phosphorylation triggers subsequent ubiquination. As a generalization, almost every step in the cell cycle involves ubiquination followed by a phosphorylation that leads to the destruction of a cell cycle regulator. In the NF κb pathway, phosphorylation leads to the degradation selectively of the inhibitor of NF κb and IκB. And there would probably be 30 to 40 examples, which we could cite, where these are linked. There are even now a couple of cases where a kinase and a ubiquitin ligase reside in the same molecule. 22

23 So, I think if you were to try designing the cell, you would think of phosphorylation dephosphorylation and ubiquination as different types of ways of regulating pathways with different time courses. But their mechanisms are definitely talking to each other. Excellent. I have a question here on the number of E3s and I understand there s something like 650 E3 ubiquitin ligases that have been identified or identified at least through genomics. And this viewer is asking, how redundant are the E3s and how difficult is it to find an E3 that recognizes a specific substrate. Dr. Gregory Korbel: I think that s an outstanding question, and we re really just beginning to get to the stage where we have the tools to begin to address that. As I ve mentioned earlier, one of the tremendous bottlenecks in this line of research has been the identification of substrates. And we really are just now beginning to piece that together. Out of the 650 or more E3 ubiquitin ligases, which are known, there are well fewer than a hundred substrates. And the proteome is clearly much, much larger than that. So, I think, there is diversity in E3s. There are multiple families of E3s. many of which are modular and mixes as multicomponent complexes, and that certainly adds to the challenge of identifying. There probably are proteins, which are recognized by multiple E3s and there are most certainly things that are going to be very specific to a given E3 as well. Dr. Paul Andrews: Dr. Paul Andrews: I think just to add to that quickly. I think 650 is probably a low number. If you start thinking about the modularity of the SCF complex, the VCD complexes, these are all multi subunit complexes of ubiquitin ligases that can bring in a number of different substrate recognition proteins. You start adding all of those together and then on top of that add the fact that the potential is there that each one of these can interact with a different E2. Uh hum. So you start adding those numbers and it becomes exponential as opposed to simply adding up the numbers. I think 650, you know, when all is said and done is probably going to be a low number. Dr. Alfred Goldberg: May I just add a point, which maybe to get back to the viewer s question, typically, there are many, many E3s. If you knock them out or reduce 23