Posts by moody

1) Message boards : Rosetta@home Science : Design of protein-protein interfaces (Message 76584) Posted 3 Apr 2014 by moody Post: Tom, I apologize for the long delay in getting back to you. The top MB17 variants bind Mdm4 (Mdmx) or Mdm2 tightly enough to yank them out of human cells and trigger the self-destruction pathway (p53 apoptosis pathway) in colon cancer cells. We're now looking at other types of cancer cells. We're now working on making the MB17 variants bind more tightly to Mdm4 or Mdm2. We're also studying how the p53 pathway works using the MB17 variants. The work is on-going so we don't have the full story yet. MB17 and its variants were intended to aid as cancer research tools and we're cautiously optimistic that they'll work to that end. We're hoping that the things we learn will fuel the development of new kinds of cancer drugs and treatment strategies. You likely have recently seen Rosetta@Home jobs with either Mdm4, Mdm2, or MB17 in the name. These represent ongoing efforts to design improved Mdm4 and Mdm2 binding proteins using new tools in the Rosetta software suite. Once again I want to give a big thank-you to you and all of our Rosetta@Home contributors! _______ [quote]To all of our wonderful Rosetta@Home contributors, Here is an update about the p53-Mdm4 project: Using Rosetta@Home, we identified a set of 14 proteins that could be modified to stick to Mdm4 while ignoring Mdm4. We synthesized these proteins, tested them, and identified one that sticks to Mdm4 about 75 times better than it sticks to Mdm2. We sent this protein, called Mdm4 Binder 17 (MB17) to St. Jude's Childrens Research Hospital so the cancer experts could test it in cancer cells. So far, they have confirmed that MB17 strongly prefers to stick to Mdm4 over Mdm2 (about 170 times better, in their experiments). They also found that MB17 works correctly inside living cells, a major milestone for a designed protein. In the mean time, we've created new versions of MB17 that preference Mdm4 even more (about 370 times more than Mdm2), some that pinch-hit and prefer Mdm2 instead of Mdm4 (about 120 times more than Mdm4), and some that like both Mdm4 and Mdm2 equally well. The folks at St. Judes are starting to work with these improved variants as well. This is the first time that the cancer research community has ever had a tool to knock out just Mdm4 while leaving Mdm2 alone, so naturally, the folks at St. Jude's are pretty excited. They are gearing up to test MB17 in real cancer cells so we'll keep our fingers crossed. We'll keep you updated as new developments arise. I'm sorry for the long delays between posts. Things are pretty busy around here. For those of you seeing this thread for the first time, I've re-posted below what I posted previously about this topic: Project 1: The first of these interactions involves a protein called p53 and another called Mdm4. p53 is a communication hub in our cells, used to translate information about unwanted DNA mutations into an effective response by the cell. The activity of p53 is modulated by a pair of other proteins, Mdm4 and Mdm2, which act to shut off p53 when all is well. Cancer cells depend on DNA mutations to stay alive and so they find ways to shut off p53, either by reducing the amount available p53 or by expanding the amount of available Mdm4 or Mdm2. that way p53 doesn't rat out mutations that the cancer might need to survive. Scientists have spent a lot of time trying to understand how Mdm4 and Mdm2 work. They do this by shutting off Mdm2 and Mdm4 with drugs. There are even drugs that will shut off just Mdm2. There are no drugs, however, that will shut off just Mdm4. So I'm trying to make one. This is a tricky process since Mdm2 and Mdm4 are very similar, making it hard for proteins to tell them apart. I aim to use Rosetta@home to design proteins that will attach to Mdm4 while ignoring Mdm2. I hope to provide a research tool to scientists that work on Mdm4, Mdm2 and p53, allowing them to better understand how this critical communication hub works. I hope that this leads to new cancer treatments. So far, I've used Rosetta@home to design 26 proteins that should attach to Mdm4 but not Mdm2. Many of the designed proteins do stick to Mdm4, but unfortunately, they also stick to Mdm2. I am working on a third round of design using a new approach that I hope will work. For more info about the proteins involved in this project, please visit the links below: They are gearing up to test MB17 in real cancer cells so we'll keep our fingers crossed. We'll keep you updated as new developments arise. Any news if the MB17 worked on the cancer cells?
2) Message boards : Rosetta@home Science : Design of protein-protein interfaces (Message 75771) Posted 18 Jun 2013 by moody Post: To our awesome Rosetta@Home contributors: At the beginning of 2012 we told you that it looked like eight designed proteins stuck to EED as desired. Further experiments showed us that of our eight initial hits, only two were real, and those two proteins were too weak to be improved with laboratory techniques. We've gone back to the drawing board, using more aggressive modeling techniques to design better binding proteins to mimic Ezh2. It turns out that no known protein structure is similar enough to the Ezh2 binding helix to allow us to simply transplant key amino acids from Ezh2 to a new host protein, as we tried before. What were doing now is making new proteins from scratch that have the exact shape that we need to mimic Ezh2. The "EED" runs you'll now see on Rosetta@Home are structure prediction of designs to see if they have the desired curvature when Rosetta folds them up. Thanks again for all of your donated computer time! For those new to this thread/message board, here is what I wrote previously about the EED project: The second of these interactions involve a protein called EED and another called Ezh2. If you think about DNA as a ladder, then each of our cells has about six billion rungs worth of ladder. In order to keep all of that DNA from getting hopelessly tangled, our cells keep it on little spools, called histones, when not in use. Think of this like a massive film archive. It turns out that there is a lot of DNA that never gets used, so the cells put special flags on those spools (histones) that say something like "never use this section of DNA". EED is a large protein that attaches to those flags and brings along Ezh2, a "flagging machine" for the ride. EED makes sure that Ezh2 adds flags to histones that are supposed to get them, and not to histones carrying DNA the cell actually wants to use. Some cancer cells reduce the amounts of EED or Ezh2 to prevent flagging of DNA regions that they want to use to take over our bodies. Other cancer cells expand the amounts of EED or Ezh2 to flag DNA regions that are getting in the way. Scientists are working hard to better understand how EED, Ezh2, and friends work in normal cells and what goes wrong in cancer cells. Some are trying to develop drugs that prevent Ezh2 from attaching to EED. This means that the drugs have to stick to EED more tightly than Ezh2. Although Ezh2 attaches relatively loosely to a large patch on the surface of EED, their aren't any drugs yet available that do what we want. I'm trying to make proteins that will do the same job. I used Rosetta@home to design a set of proteins that mimic Ezh2 in order to block it from attaching to EED. Just to give you a sense of how much computing I need for a project like this, I submitted just under two million work units to Rosetta@home, and donor's computers ran my protocol just under a billion times to give me about 54 designs to look at, from which fourteen were suitable for testing. This took about a month to run on Rosetta@home. From initial experiments, it looks like eight of the designs stick to EED, and one in particular sticks three times better than the Ezh2 found in our cells. Now I'm working to improve the best design at the lab bench and hope to send it to co-workers for testing in living cells. There's still a lot of work to be done to make sure that everything is working right with this design, but I want to give a big thank-you to all of you who donated your computer time to make this possible! For more information about EED, Ezh2, and related proteins, please visit: <http://en.wikipedia.org/wiki/EED> <http://en.wikipedia.org/wiki/SUZ12> <http://en.wikipedia.org/wiki/EZH2> <http://en.wikipedia.org/wiki/PRC2> <http://en.wikipedia.org/wiki/Polycomb-group_proteins> Work units for this project carried the word "EED" in their name.
3) Message boards : Rosetta@home Science : Design of protein-protein interfaces (Message 75635) Posted 20 May 2013 by moody Post: To all of our wonderful Rosetta@Home contributors, Here is an update about the p53-Mdm4 project: Using Rosetta@Home, we identified a set of 14 proteins that could be modified to stick to Mdm4 while ignoring Mdm4. We synthesized these proteins, tested them, and identified one that sticks to Mdm4 about 75 times better than it sticks to Mdm2. We sent this protein, called Mdm4 Binder 17 (MB17) to St. Jude's Childrens Research Hospital so the cancer experts could test it in cancer cells. So far, they have confirmed that MB17 strongly prefers to stick to Mdm4 over Mdm2 (about 170 times better, in their experiments). They also found that MB17 works correctly inside living cells, a major milestone for a designed protein. In the mean time, we've created new versions of MB17 that preference Mdm4 even more (about 370 times more than Mdm2), some that pinch-hit and prefer Mdm2 instead of Mdm4 (about 120 times more than Mdm4), and some that like both Mdm4 and Mdm2 equally well. The folks at St. Judes are starting to work with these improved variants as well. This is the first time that the cancer research community has ever had a tool to knock out just Mdm4 while leaving Mdm2 alone, so naturally, the folks at St. Jude's are pretty excited. They are gearing up to test MB17 in real cancer cells so we'll keep our fingers crossed. We'll keep you updated as new developments arise. I'm sorry for the long delays between posts. Things are pretty busy around here. For those of you seeing this thread for the first time, I've re-posted below what I posted previously about this topic: Project 1: The first of these interactions involves a protein called p53 and another called Mdm4. p53 is a communication hub in our cells, used to translate information about unwanted DNA mutations into an effective response by the cell. The activity of p53 is modulated by a pair of other proteins, Mdm4 and Mdm2, which act to shut off p53 when all is well. Cancer cells depend on DNA mutations to stay alive and so they find ways to shut off p53, either by reducing the amount available p53 or by expanding the amount of available Mdm4 or Mdm2. that way p53 doesn't rat out mutations that the cancer might need to survive. Scientists have spent a lot of time trying to understand how Mdm4 and Mdm2 work. They do this by shutting off Mdm2 and Mdm4 with drugs. There are even drugs that will shut off just Mdm2. There are no drugs, however, that will shut off just Mdm4. So I'm trying to make one. This is a tricky process since Mdm2 and Mdm4 are very similar, making it hard for proteins to tell them apart. I aim to use Rosetta@home to design proteins that will attach to Mdm4 while ignoring Mdm2. I hope to provide a research tool to scientists that work on Mdm4, Mdm2 and p53, allowing them to better understand how this critical communication hub works. I hope that this leads to new cancer treatments. So far, I've used Rosetta@home to design 26 proteins that should attach to Mdm4 but not Mdm2. Many of the designed proteins do stick to Mdm4, but unfortunately, they also stick to Mdm2. I am working on a third round of design using a new approach that I hope will work. For more info about the proteins involved in this project, please visit the links below:
4) Message boards : Rosetta@home Science : Design of protein-protein interfaces (Message 73691) Posted 24 Aug 2012 by moody Post: Computationally-designed Wnt Surrogate Protein Wnt protein is widely recognized as a crucial component of vertebrate development. For more than three decades, scientists have sought to understand the structure of this important molecule. Unfortunately, obtaining a detailed understanding of Wnt's structure has proven to be quite difficult. After much work, a research group led by Dr. K. Christopher Garcia at Stanford University published the structure of Wnt bound to its target protein in June 2012. This binding event is partially facilitated by a fatty acid, an addendum to Wnt that is known to complicate molecular structure determination. Wnt protein is secreted into the space around growing cells. It then binds to its target (Frizzled protein) on the surface of some of these cells. The attachment of Wnt to Frizzled leads to the transmission of a signal into the cell, which alters the development and physiological behavior of that cell by changing the way that the cell accesses the information in its DNA. This signaling event is modified by a number of supplementary proteins in the same pathway. Our current project aims to replace naturally occurring Wnt with a surrogate protein through protein engineering methods. We first look at the structure of Wnt's target protein and use computer models that rely on the Baker Lab's Rosetta computational design technology to determine chemically favorable binding locations. We then look at how we might combine the mixture of possible binding sites into an optimal binding pattern. Finally, we use the distributed computing abilities of the BOINC network to attempt to find a previously-studied protein that can be seeded with our binding pattern. In a successfully engineered project, the newly created protein will show its ability to bind to the target (Frizzled in this case) during validation tests. In parallel with our standard techniques, which already rely heavily on the CPU time of our Rosetta@home participants, we have submitted the structure of the target and a potential binder to FoldIt players. FoldIt will allow people to interact directly with the Rosetta scoring algorithms, allowing the binding region to be custom-designed with the help of real-time feedback. The creation of a successful Wnt-surrogate binder will signify an important advance in our ability to quickly employ emerging scientific data to facilitate the clinically-focused goals of our team of molecular engineers. Binding to Frizzled should allow us to interrupt the Wnt signaling pathway in a way that will be immediately useful as a tool for the many laboratories that are focused on human development. Looking farther into the future, control of the Wnt signaling pathway may allow us to limit the growth of Wnt-mediated tumors and may even prove useful in tissue engineering. Through the efforts of the scientists in the Baker Lab, our partners who dedicate their computing time to Rosetta@home, and FoldIt players, we will begin testing the preliminary designs for a Wnt surrogate at our principal laboratory in Seattle, Washington in late August 2012.
5) Message boards : Rosetta@home Science : Design of protein-protein interfaces (Message 72164) Posted 17 Jan 2012 by moody Post: And here is the third of the projects I mentioned in posts 72162 and 72155: Project 3: The third of these interactions involve a protein called RhoA and another protein called Dbs. Most of the cells in our bodies have the ability to crawl around on command. This is really important when we develop as embryos. Cells are crawling everywhere to get into the right spot and morph us from something that looks like an alien into something that looks like a baby. As adults, must of our cells have stopped crawling around and settled down to do their respective jobs. If you have ever cut yourself and noticed that the cut got smaller and smaller as it healed, that's because the skin cells on the edges of the cut crawled into the gap to seal it up. Cells have rigid skeletons to give them shape and keep their insides organized, so in order the crawl around, they have to constantly rearrange their skeletons as they go. Just imagine the information processing and communication that has to happen inside the cell for this to happen in an organized manner. Imagine a circus tent with a thousand people inside it and they all decide to move the tent a mile away without taking it down or anyone leaving the tent. There are lots of protein-protein interactions required for this to work right in our cells. One of these is a protein switch called RhoA. RhoA can be either in the "on" position or the "off" position. When a cell wants to crawl around, it uses Dbs to switch RhoA to the "on" position so that the cell's skeleton will be rearranged faster. When cells don't want to move around, they use proteins called RhoGAPs to turn RhoA "off" so that the cell's skeleton is rearranged more slowly. Since cancer cells grow like crazy, eventually things become crowded where they live and so some of the cancer cells decide to strike out on their own, colonizing new parts of our bodies. Doctors call this "metastasis" and it's bad news for cancer patients. In order to start crawling to a new home, a cancer cell speeds up rearrangement of the it's skeleton, either by expanding the amounts of RhoA or Dbs in the cell or by reducing the amounts of RhoGAPs so that they aren't around to turn off RhoA. RhoA is a very flexible protein and no one has been able to develop a drug that will keep RhoA turned "off" by preventing Dbs from attaching and switching it "on". I'm trying to make proteins that will attach to RhoA and prevent Dbs from switching it "on". This is a tricky venture, since RhoA is very, very flexible and attaches only loosely to Dbs. I used Rosetta@home to design a set of 16 proteins to do the job, but none of these stuck to RhoA when tested. In the coming months I'll use Rosetta@home to try a different design strategy that I think will work. If successful, these proteins would be the first RhoA inhibitors available and should provide scientists a new tool to better study how RhoA works and how cancer cells take control of it. Work units for this project will contain the word "RhoA" in their name. Thanks again for all of your donated computer time. You're help makes these projects possible!
6) Message boards : Rosetta@home Science : Design of protein-protein interfaces (Message 72162) Posted 17 Jan 2012 by moody Post: As promised, here is the continuation of the story begun in message 72155: Project 2: The second of these interactions involve a protein called EED and another called Ezh2. If you think about DNA as a ladder, then each of our cells has about six billion rungs worth of ladder. In order to keep all of that DNA from getting hopelessly tangled, our cells keep it on little spools, called histones, when not in use. Think of this like a massive film archive. It turns out that there is a lot of DNA that never gets used, so the cells put special flags on those spools (histones) that say something like "never use this section of DNA". EED is a large protein that attaches to those flags and brings along Ezh2, a "flagging machine" for the ride. EED makes sure that Ezh2 adds flags to histones that are supposed to get them, and not to histones carrying DNA the cell actually wants to use. Some cancer cells reduce the amounts of EED or Ezh2 to prevent flagging of DNA regions that they want to use to take over our bodies. Other cancer cells expand the amounts of EED or Ezh2 to flag DNA regions that are getting in the way. Scientists are working hard to better understand how EED, Ezh2, and friends work in normal cells and what goes wrong in cancer cells. Some are trying to develop drugs that prevent Ezh2 from attaching to EED. This means that the drugs have to stick to EED more tightly than Ezh2. Although Ezh2 attaches relatively loosely to a large patch on the surface of EED, their aren't any drugs yet available that do what we want. I'm trying to make proteins that will do the same job. I used Rosetta@home to design a set of proteins that mimic Ezh2 in order to block it from attaching to EED. Just to give you a sense of how much computing I need for a project like this, I submitted just under two million work units to Rosetta@home, and donor's computers ran my protocol just under a billion times to give me about 54 designs to look at, from which fourteen were suitable for testing. This took about a month to run on Rosetta@home. From initial experiments, it looks like eight of the designs stick to EED, and one in particular sticks three times better than the Ezh2 found in our cells. Now I'm working to improve the best design at the lab bench and hope to send it to co-workers for testing in living cells. There's still a lot of work to be done to make sure that everything is working right with this design, but I want to give a big thank-you to all of you who donated your computer time to make this possible! For more information about EED, Ezh2, and related proteins, please visit: <http://en.wikipedia.org/wiki/EED> <http://en.wikipedia.org/wiki/SUZ12> <http://en.wikipedia.org/wiki/EZH2> <http://en.wikipedia.org/wiki/PRC2> <http://en.wikipedia.org/wiki/Polycomb-group_proteins> Work units for this project carried the word "EED" in their name. I'll post more about the third project shortly.
7) Message boards : Rosetta@home Science : Design of protein-protein interfaces (Message 72161) Posted 17 Jan 2012 by moody Post: Good call. Work units involving the design of proteins to bind to Mdm4 will contain the word "Mdm4" in their name.
8) Message boards : Rosetta@home Science : Design of protein-protein interfaces (Message 72155) Posted 17 Jan 2012 by moody Post: In response to recent requests for an update on what we are doing with your donated computer time, here goes: The communication networks inside every one of our cells depend in many cases on proteins interacting with one another. When these communication networks go awry, cancer often results. I'm using Rosetta to design proteins that will interfere with three protein-protein interactions often involved in cancer. I'll tell you about the first project below, and the others shortly. Project 1: The first of these interactions involves a protein called p53 and another called Mdm4. p53 is a communication hub in our cells, used to translate information about unwanted DNA mutations into an effective response by the cell. The activity of p53 is modulated by a pair of other proteins, Mdm4 and Mdm2, which act to shut off p53 when all is well. Cancer cells depend on DNA mutations to stay alive and so they find ways to shut off p53, either by reducing the amount available p53 or by expanding the amount of available Mdm4 or Mdm2. that way p53 doesn't rat out mutations that the cancer might need to survive. Scientists have spent a lot of time trying to understand how Mdm4 and Mdm2 work. They do this by shutting off Mdm2 and Mdm4 with drugs. There are even drugs that will shut off just Mdm2. There are no drugs, however, that will shut off just Mdm4. So I'm trying to make one. This is a tricky process since Mdm2 and Mdm4 are very similar, making it hard for proteins to tell them apart. I aim to use Rosetta@home to design proteins that will attach to Mdm4 while ignoring Mdm2. I hope to provide a research tool to scientists that work on Mdm4, Mdm2 and p53, allowing them to better understand how this critical communication hub works. I hope that this leads to new cancer treatments. So far, I've used Rosetta@home to design 26 proteins that should attach to Mdm4 but not Mdm2. Many of the designed proteins do stick to Mdm4, but unfortunately, they also stick to Mdm2. I am working on a third round of design using a new approach that I hope will work. For more info about the proteins involved in this project, please visit the links below: <http://en.wikipedia.org/wiki/MDM4> <http://en.wikipedia.org/wiki/Mdm2> <http://en.wikipedia.org/wiki/P53> Thanks for lending us your spare computer time. Projects like this one require so much computational time that they would be impossible without you. I'll post more about the other two projects at a later date.
9) Message boards : Number crunching : minirosetta 2.17 (Message 70030) Posted 13 Apr 2011 by moody Post: In response to Message 70021: "A couple of new protein interface design tasks failing immediately with a computation error on Mac. Sample: Task 414128879 (dck_rhoA_rhoA_2nr7_final_ProteinInterfaceDesign_11Apr2011_25012_119_0) ERROR: Option matching -docking:no_filters not found in command line top-level context" This was due to a Rosetta option that was recently renamed on our end but not in the version of Rosetta currently on Boinc. It should be fixed now. We apologize for any inconvenience.
10) Message boards : Rosetta@home Science : Design of protein-protein interfaces (Message 68860) Posted 22 Dec 2010 by moody Post: For those you you asking about EED: See <http://boinc.bakerlab.org/forum_thread.php?id=4477&nowrap=true#66689> for a description of this project. If you have other questions don't hesitate to ask! Thanks again for all of your help! --James
11) Message boards : Rosetta@home Science : Design of protein-protein interfaces (Message 66689) Posted 24 Jun 2010 by moody Post: Hello, My name is James Moody and I am a new graduate student in the Baker lab. I am working on protein-protein interface design and am excited to have the help of all of the participants of Rosetta@home! Right now I am working on designing a protein to bind to a regulatory molecule called EED. EED works with other proteins in our cells to control which parts of our DNA will be used to control the cell and which parts will be silenced (turned off). EED is thought to work by bringing other proteins together and is part of a larger protein machine called the Polycomb Repressive Complex 2 (PRC2). PRC2 helps to ensure, for example, that we have the right number of arms and legs and that they are in the right place on our bodies (by controlling our Hox genes). Scientists are working to better understand how EED works to control the activity of our DNA and its link to things like stem cells, development, and cancer. To do this, we need a way to interrupt the normal function of EED. This is especially difficult since there are currently no drugs that block EED function. An engineered protein would be able to prevent EED from sticking to one of its binding partners (histone tails) and could be turned on and off at will by the researcher. It is hoped that such a protein would allow researchers to carry out new experiments on EED that were impossible before. Engineering this novel interaction with EED into another protein requires computationally screening through as many as possible of a billion possibilities, evaluating each protein for how tightly it sticks to EED, and then redesigning the new protein to stick even better. Such a task would be impossible without the help of Rosetta@home participants! Thank you so much for lending yourselves to participate in this project. Things that I send to rosetta@home are tested first on our machines and then on a subset of Rosetta@home participants to ensure that they don't cause any problems for you. Please don't hesitate to ask if you have questions, feedback, or see errors. If you would like to learn more about EED or related topics, the following webpages might be helpful! http://en.wikipedia.org/wiki/Polycomb-group_proteins http://en.wikipedia.org/wiki/EED http://en.wikipedia.org/wiki/Histones http://en.wikipedia.org/wiki/Nucleosome http://en.wikipedia.org/wiki/Hox_genes --James