By Jovana Drinjakovic
University of Toronto scientists have uncovered more than 300 drug targets in cancer, attracting interest from the pharmaceutical industry looking to develop more precise treatments. Led by Professor Igor Stagljar of U of T’s Donnelly Centre, the study maps interactions between receptor tyrosine kinases (RTKs) and protein tyrosine phosphatases (PTPs) in humans, which can lead to cancer when their functions are disrupted. The highly anticipated study will be featured on the cover of the journal Molecular Cell, available in print on January 19.
Most cancer patients are treated with punishing chemotherapy drugs that have serious side-effects. In the last 15 years, a new generation of "smart" cancer drugs has been developed such as Gleevec, which effectively cures some forms of leukemia. These drugs are designed to target cancer cells with needle-like precision to avoid harming tissue that’s healthy. They do this by blocking proteins called kinases, which include receptor tyrosine kinases (RTKs) that control cell growth. RTKs are often mutated in cancer. However, the existing drugs block only a fraction of RTKs because these proteins have features that have made them notoriously hard to study.
Senior research associate Dr. Zhong Yao was able to carry out the largest study of RTKs to date by mapping their physical interactions with PTPs using methods previously developed in Stagljar’s lab.
“We tested interactions between almost all 58 RTKs and 144 PTPs that exist in human cells. Our map reveals new and surprising ways in which these proteins work together. These insights will help us better understand what goes wrong in cancer in order to develop more effective treatments,” said Stagljar, who is also a professor in U of T’s molecular genetics and biochemistry departments.
Lodged inside the cell’s outer envelope, or membrane, RTKs receive signals from the outside world—a hormone, for example— telling the cell to grow and divide. Normally, their activity is controlled by PTPs, which bind the RTKs and shut them down. This prevents sustained cell division that could lead to cancer.
The RTK’s place in the cell membrane is critical for their function, but it is also what has made them such a tough nut to crack. Traditional methods haven’t been able to capture the often short-lived physical interactions between RTKs and PTPs because the surrounding membrane has to be dissolved, which changes the proteins’ behaviour. Stagljar bridged this gap by developing MYTH and MaMTH, technologies designed precisely for measuring such fleeting interactions between membrane proteins in their natural setting.
The resulting map charts out more than 300 interactions between RTKs and PTPs in human cells, each a potential way to fight cancer. The findings have attracted attention of major pharmaceutical companies, including the pharma giant Genentech, which could lead to future collaborations in drug development.
Stagljar worked with two leading experts in PTP biology: Professor Anne-Claude Gingras of the Lunenfeld-Tanenbaum Research Institute and U of T’s Department of Molecular Genetics, and Professor Benjamin Neel of New York University, who was formerly with U of T and the University Health Network’s Princess Margaret Cancer Center.
“We wanted to show that these two assays we developed in our lab – MYTH and MaMTH – are suitable for studying these two important classes of proteins on such a large scale. The resulting wealth of important data can be used to develop new therapies against various types of cancer,” said Stagljar. “Ultimately, we want to build a map of interactions with all 3,000 or so human membrane proteins, of which at least 500 have direct roles in the onset of many human diseases. This will keep us busy,” he added.
"CRISPR genome editing is quickly revolutionizing biomedical research, but the new technology is not yet exact. The technique can inadvertently make excessive or unwanted changes in the genome and create off-target mutations, limiting safety and efficacy." More
By Jovana Drinjakovic
The study could pave the way for more meaningful interpretation of personal genomes.
Toronto scientists have discovered that the largest group of human proteins, which work as genome gatekeepers to control gene activity, are even more diverse in their roles than previously thought. The finding expands our understanding of how proteins “read” the DNA and could lead to a more accurate interpretation of individual genomes.
Donnelly Centre teams, led by Professor Timothy Hughes and University Professor Jack Greenblatt, have shown that proteins called C2H2-zinc fingers (C2H2-ZF) can control gene activity in new and surprizing ways. Reporting in the December issue of Genome Research, the researchers also reveal DNA binding sites for more than a hundred C2H2-ZFs as part of an ongoing effort to decode genome sequences that do not code for genes.
Despite being the largest group of human proteins—counting 700 members—the C2H2-ZFs are poorly understood partly because their sheer abundance and diversity make them hard to study. Yet knowing how they work is important because they help orchestrate gene activity. Of 20,000 human genes, only a subset is active in the cell at any given time. This subset determines if the cell will, say, build blood, or the brain or go haywire to become cancer.
The C2H2-ZF proteins work by directly binding the DNA to control the genes nearby. Named after their finger-like structures that, aided by zinc ions, clasp the DNA, C2H2-ZFs have previously been thought to act by stifling a wide range of genes. In a previous study that included about 40 C2H2-ZFs, the team showed that each protein recognized a unique DNA snippet as its landing site in the genome, raising the possibility that the rest of the group could be just as diverse.
This was indeed confirmed in the present study in which the teams mapped DNA binding sites, most of which were unique, this time for 131 C2H2-ZF proteins. But they also uncovered a whole new way in which the C2H2-ZF proteins can be regulated to vastly expand their job repertoire in the cell.
In addition to binding the DNA, it turned out that each C2H2-ZF can partner with a motley of other proteins that could potentially tweak its ability to switch genes on and off in a unique way. The finding upended the previous thinking in which C2H2-ZF proteins were seen as limited in their ability to bind other proteins—half of them were thought to interact with a single protein that helps them gag target genes, while the rest lack the usual molecular features that help proteins contact one another.
“Our key finding is that there’s almost as much diversity in the protein-protein interactions as there is in the DNA binding sequences. It tells us that the way the C2H2-ZF proteins work is almost certainly more complicated than we would have expected,” said Hughes, who is also a professor in U of T’s department of molecular genetics and a fellow of the Canadian Institute for Advanced Research (CIFAR).
The kinds of proteins that C2H2-ZFs interact with suggest that their roles go beyond clamping down on genes and may even act to turn genes on or help package DNA inside the cell.
The study also shines light on how the C2H2-ZF evolved to become the largest and most diverse group of proteins we have. The DNA sequences that C2H2-ZF proteins recognize look a lot like they had come from viruses, some of which plagued our mammalian ancestors as long as 100 million years ago. This kind of viral DNA has been called “selfish DNA” because it spreads by inserting itself randomly in a host’s genome.
It is thought that the C2H2-ZF proteins evolved to shut down this selfish DNA, their legion expanding to keep up with new intruders. Once the viral DNA was squashed for good, the C2H2-ZF proteins were able to take on new roles in shutting down mammalian genes. And now, this new data suggest that the C2H2-ZF proteins branched out even more than previously thought to acquire wholly unexpected functions by binding to other proteins.
Knowing how C2H2-ZFs work will give scientists a better handle on predicting which genes they control and how this may relate to disease. So far, mass genome sequencing studies have fallen short from being able to tell one’s risk of common diseases, such as cancer or diabetes, because we still don’t know enough about the meaning of individual differences between genomes.
“Even today, 15 years after the human genome was sequenced, if you give any piece of DNA to a geneticist and ask them what this does, generally they are unable to tell you that. But the more we learn about how human proteins recognize the DNA and what they do, the better our ability will be to interpret genome sequences and say what the significance of the variants is,” said Hughes.
U of T Researchers Rapidly Identify New Drug Target and Develop Antibodies to Kill Pancreatic Cancer Cells
By Jef Ekins
Researchers at the University of Toronto have developed a process that dramatically cuts the amount of time it takes to create new cancer treatments. Using a breakthrough technology, their study, published this week in Nature Medicine, identified a new potential drug target in a class of pancreatic cancer, and unveiled a new treatment option that exploits genetic faults to destroy cancer cells.
Professors Jason Moffat and Sachdev Sidhu from the Donnelly Centre and the department of molecular genetics, along with Professor Stephane Angers from the Leslie Dan Faculty of Pharmacy, made this discovery using the cutting-edge CRISPR-Cas9 genome editing technology.
Using this revolutionary tool, the teams probed the function of every single gene expressed by pancreatic cancer cells to determine that one of the receptors (Frizzled-5) is essential for the growth of mutant pancreatic cancer cells. Normally, the signaling pathways activated by Frizzled-5 tell cells when to divide, what types of cells to become, and when they should die. When mutated or deregulated, however, they can initiate tumour growth.
"This is the first time that we are able to identify bona fine genetic weaknesses in cancer cells that we can target with drugs, which will not harm healthy tissue," said Moffat.
Having identified the key role that the Frizzled-5 receptor plays in promoting pancreatic cancer growth, the team rapidly developed an antibody drug to inhibit the growth of these cells. The study showed that the antibody proved highly effective in killing the cancer cells in patient-derived samples and shrank tumours in mice without damaging the surrounding healthy cells.
Leveraging the Donnelly Centre’s state-of-the-art platform for custom antibody design, the team was able to create a targeted antibody in months – a fraction of the time it would normally take to develop a safe and effective treatment for a specific cancer.
"Our technology allows us to quickly develop a drug that's tailored to a particular kind of cancer and ready to be tested on people. In this study we show that the approach works for a type of pancreatic cancer, but this is only the tip of the iceberg," said Sidhu.
As part of this study, the team also explored the role of this receptor in colorectal cancer, a form of cancer that shares common features with pancreatic cancer. The results of this study indicate that Frizzled-5 may be a factor across multiple cancer types, broadening the potential use of anti-Frizzled-5 antibodies as a targeted cancer therapy.
“Ultimately, this study revealed genetic vulnerabilities in pancreatic cancer cells that could be exploited through the development of new targeted antibodies to inhibit tumor growth,” said Angers. “By targeting the exact signaling circuit activated in these tumors, these rapidly developed antibodies have shown considerable promise as a cancer treatment. Moreover, the state-of-the-art antibody development platform developed at U of T is a transformational leap forward in our ability to rapidly create exciting new treatments to combat various cancers.”
By Jovana Drinjakovic
As a protein engineer, Dr. Wei Zhang gives old molecules new tricks. And now, he’s transformed a single human protein into a virus-crushing arsenal that could lead to long-sought treatments for deadly infections.
This week, Zhang received the Mitacs Award for Outstanding Innovation, for his work on creating molecular antidotes against viruses that cause Middle East Respiratory Syndrome (MERS) and Crimean-Congo Hemorrhagic Fever (Congo Fever). His patent-pending research was crucial to the launch of a new Toronto-based company called Ubiquitech, which will further commercialize his work so it can be used in a clinical setting.
As a postdoctoral research fellow in Professor Jason Moffat’s group in the Donnelly Centre, Zhang already holds a competitive Elevate Fellowship from Mitacs, a nonprofit supporting innovation across the public and private sectors. The fellowship enabled Zhang to start thinking about commercializing his research through a collaboration the with the Centre for Commercialization of Antibodies and Biologics (CCAB), the industry partner for his fellowship, located at the U of T.
“Wei has done a phenomenal job of applying a cutting-edge new technology to the pressing issue of emerging pathogenic viruses. It’s a great example of how an investment by Canada in basic research and talented young scientists can lead to real impacts on human health,” says Dr. Sachdev Sidhu, CEO of CCAB.
After completing a doctorate with a U of T Professor Daniel Durocher, Zhang joined Moffat and Sidhu’s labs to learn the ropes of protein engineering. The two teams had previously developed a powerful technology to quickly create synthetic proteins that could be used as research reagents, or developed further into drugs.
Although Zhang’s work revolves around a single protein, called ubiquitin, its applications are far-reaching. Named after its pervasive presence in every cell on earth, ubiquitin works by attaching to other proteins to help relay signals telling the cell to grow or fight infections, for example. Encoded by genes, proteins do most of the work in the cell. They are built from amino acids, which are stitched together based on the DNA blueprint. Using molecular tricks, scientists can change how a protein behaves by changing its DNA sequence.
And so, through subtle tweaks, Zhang has turned a lone, naturally occurring ubiquitin into a set of tools—synthetic ubiquitin variants (UbV)—which allow him to manipulate the proteins that ubiquitin normally binds to. For example, a ubiquitin variant may spur the activity of the other protein, or it may take it out of action completely. But each variant has a unique target, allowing Zhang to control protein activity with unrivalled precision.
Being able to thwart a protein is particularly useful when dealing with harmful molecules, like those made by bacteria and viruses, for example.
“Ubiquitin-dependent signalling is important in the immune response, and a lot of viruses encode proteins that bind human ubiquitin, which allows them to topple the body’s defense mechanism. One of them is MERS, a respiratory virus similar to SARS that caused a global epidemic in 2002. 14 years later, nothing has come out of clinical trials. While vaccines are being developed for many viruses, there is no treatment in sight for people who are infected,” says Zhang.
MERS emerged in Saudi Arabia in 2012, and it kills almost 40 per cent of those who become infected, making it even more dangerous than SARS. Zhang has engineered a ubiquitin variant which blocks MERS’ ability to evade the immune response. “When we treat the cells infected with MERS with the ubiquitin variant, we can kill the virus in two days,” says Zhang.
The list of anti-viral ubiquitins could become long. Zhang has already created a variant that’s effective against Congo Fever virus, which causes internal bleeding and kills almost half of those infected. But what’s most exciting about Zhang’s approach is that it can be applied to any viral protein that binds ubiquitin. “We are able to quickly—in less than one month—generate inhibitors for any ubiquitin-binding proteins in the virus,” says Zhang. This means that Zhang’s work could also be applied to viruses such as SARS, Zika, and Ebola, as well as to preventing viral damage to food crops and animals.
“Wei has fully captured the potential of protein engineering technology with his research. He is poised to turn basic science into applications that could help people,” says Moffat.
By Jovana Drinjakovic
The study paves the way for understanding how some people stay healthy despite having disease-causing mutations.
We usually think of mutations as errors in our genes that will make us sick. But not all errors are bad, and some can even cancel out the fallout of those mutations known to cause disease. While little has been known about this process — called genetic suppression — that will soon change as University of Toronto researchers uncover the general rules behind it.
Teams led by Professors Brenda Andrews, Charles Boone and Frederick Roth of the Donnelly Centre and the Department of Molecular Genetics, in collaboration with Professor Chad Myers, of the University of Minnesota-Twin Cities, have compiled the first comprehensive set of suppressive mutations in a cell, as reported in Science today. The four researchers are members of the Genetic Networks program of the Canadian Institute for Advanced Research. Their findings could help explain how suppressive mutations combine with disease-causing mutations to soften the blow or even prevent disease.
This curious bit of biology has only come to light as more healthy people have had their genomes sequenced. Among them are a few extremely lucky folks who remain healthy despite carrying catastrophic mutations that cause debilitating disorders, such as Cystic Fibrosis or Fanconi anemia.
How could this be?
“We don’t really understand why some people with damaging mutations get the disease and some don’t. Some of this could be due to environment, but a lot of could be due to the presence of other mutations that are suppressing the effects of the first mutation,” said Roth, who is also a Senior Scientist at Sinai Health System’s Lunenfeld-Tanenbaum Research Institute.
Imagine being stuck in a room with a broken thermostat and it’s getting too hot. To cool down, you could fix the thermostat—or you could just break a window. Genetic suppression essentially “breaks the window” to keep cells healthy despite damaging mutations. And it opens a new way of understanding, and maybe even treating, genetic disorders.
“If we know the genes in which these suppressive mutations occur, then we can understand how they relate to the disease-causing genes and that may guide future drug development,” said Dr. Jolanda van Leeuwen, a postdoctoral fellow in the Boone lab and one of the scientists who spearheaded the work.
But finding these mutations is not easy; it’s a proverbial needle in the haystack. A suppressive mutation could, in theory, be any one of the hundreds of thousands of misspellings in the DNA, scattered across the 20,000 human genes, which make every genome unique. To test them all would be impractical.
“A study like this has never been done on a global scale. And since it is not possible to do these experiments in humans, we used yeast as a model organism, in which we can know exactly how mutations affect the cell’s health,” said Van Leeuwen. With only 6,000 genes, yeast cells are a simpler version of our own, yet the same basic rules of genetics apply to both.
The teams took a two-pronged approach. On the one hand, they analyzed all published data on known suppressive relationships between yeast genes. While informative, these results were inevitably skewed towards the most popular genes — the ones that scientists have already studied in detail. Which is why Van Leeuwen and colleagues also carried out an unbiased analysis by measuring how well the cells grew when they carried a damaging mutation on its own, or in combination with another mutation. Because harmful mutations slow down cell growth, any improvement in growth rate was thanks to the suppressive mutation in a second gene. These experiments revealed hundreds of suppressor mutations for the known damaging mutations.
Importantly, regardless of the approach, the data point to the same conclusion. To find suppressor genes, we often don’t need to look far from the genes with damaging mutations. These genes tend to have similar roles in the cell — either because their protein products are physically located in the same place, or because they work in the same molecular pathway.
“We’ve uncovered fundamental principles of genetic suppression and show that damaging mutations and their suppressors are generally found in genes that are functionally related. Instead of looking for a needle in the haystack, we can now narrow down our focus when searching for suppressors of genetic disorders in humans. We’ve gone from a search area spanning 20,000 genes to hundreds, or even dozens. That’s a big step forward,” said Boone.
Press contact: Jovana Drinjakovic, +1 416 543 7820, Jovana.email@example.com
By Jovana Drinjakovic
The new map breaks away from the old way of studying genes one at a time and shows how genes interact in groups to shed light on the genetic roots of diseases.
Donnelly Centre researchers have created the first map that shows the global genetic interaction network of a cell. It begins to explain how thousands of genes coordinate with one another to orchestrate cellular life.
The study was led by U of T Professors Brenda Andrews and Charles Boone, and Professor Chad Myers of the University of Minnesota-Twin Cities. It opens the door to a new way of exploring how genes contribute to disease, with a potential for developing finely-tuned therapies. The findings are published in the journal Science on September 23.
“We’ve created a reference guide for how to chart genetic interactions in a cell. We can now tell what kind of properties to look for in searching for highly connected genes in human genetic networks with the potential to impact genetic diseases,” said Dr. Michael Costanzo, a Research Associate in the Boone lab and one of the researchers who spearheaded the study.
The study took 15 years to complete and adds to Andrews’ rich scientific legacy for which she was awarded a Companion of the Order of Canada – the highest civilian honour in the country.
Just as societies in the world are organized from countries down to local communities, the genes in cells operate in hierarchical networks to organize cellular life. If we are to understand what 20,000 human genes do, we must first find out how they are connected to each other.
Studies in yeast cells first showed the need to look farther than a gene’s individual effect to understand its role. With 6,000 genes, many of which are also found in humans, yeast cells are relatively simple but powerful stand-ins for human cells. Over a decade ago, an international consortium of scientists first deleted every yeast gene, one by one. They were surprised to find that only one in five were essential for survival. It wasn’t until the last year that advances in gene editing technology allowed scientists to tackle the equivalent question in human cells. It revealed the same answer: a mere fraction of genes are essential in human cells too.
These findings suggested most genes are “buffered” to protect the cells from mutations and environmental stresses. To understand how this buffering works, scientists had to ask if cells can survive upon losing more than one gene at a time, and they had to test millions of gene pairs. Andrews, Boone and Myers led the pioneering work in yeast cells by deleting two genes at a time, in all possible pairwise combinations, to find gene pairs that are essential for survival. This called for custom-built robots and a state-of-the-art automated pipeline to analyze almost all of the mind-blowing 18 million different combinations.
The yeast map identified genes that work together in a cell. It shows how, if a gene function is lost, there’s another gene in the genome to fill its role. Consider a bicycle analogy: a wheel is akin to an essential gene – without it, you couldn’t ride the bike. Front brakes? Well, as long as the back brakes are working, you might be able to get by. But if you were to lose both sets of brakes, you are heading for trouble. Geneticists would say that front and back brakes are “synthetic lethal,” meaning that losing both, but neither by themselves, spells doom. Synthetic lethal gene pairs are relatively rare, but because they tend to control the same process in the cell, they reveal important information about genes we don’t know much about – for example, scientists can predict what an unexplored gene does in the cell, simply based on its genetic interaction patterns.
It’s becoming increasingly clear that human genes also have one or more functional backups. So instead of searching for single genes underlying diseases, we could instead be looking for gene pairs. That is a huge challenge because it means examining about 200 million possible gene pairs in the human genome for association with a disease.
Fortunately, with the know-how from the yeast map, researchers can now begin to map genetic interactions in human cells, and even expand it to different cell types. Together with whole-genome sequences and health parameters measured by new personal devices, it should finally become possible to find combinations of genes that underlie human physiology and disease.
“Without our many years of genetic network analysis with yeast, you wouldn’t have known the extent to which genetic interactions drive cellular life or how to begin mapping a global genetic network in human cells. We have tested the method to completion in a model system to provide the proof of principle for how to approach this problem in human cells. There’s no doubt it will work and generate a wealth of new information,” said Boone, who is also a professor in U of T’s molecular genetics department, a Senior Fellow and a co-director of the Genetic Networks program at the Canadian Institute for Advanced Research (CIFAR) and holds Canada Research Chair in Proteomics, Bioinformatics and Functional Genomics.
The concept of synthetic lethality is already changing cancer treatment because of its potential to identify drug targets that exist only in tumour cells. Cancer cells differ from normal cells in that they have scrambled genomes, littered with mutations. They’re like a bicycle without a set of brakes. If scientists could find the highly vulnerable back-up genes in cancer, they could target specific drugs at them to destroy only the cells that are sick, leaving the healthy ones untouched.
August 09, 2016
By Jovana Drinjakovic
If you want to know what the Moon really looks like, you should go visit. Yet most of us will only ever know it as a shiny circle in the sky. It’s like that for biologists. When they look at molecules, they see blobs because most microscopes aren’t powerful enough to discern any structural detail.
But now, thanks to a collaboration between Mount Sinai Hospital’s Lunenfeld-Tanenbaum Research Institute (LTRI), part of Sinai Health System, and The Hospital for Sick Children (SickKids), Toronto scientists have caught a glimpse of the molecules involved in DNA repair — a process that counteracts DNA damage caused by, for example, radiation or chemicals. Published in the latest issue of Nature, the finding deepens our understanding of how cells see and respond to DNA damage and opens new avenues of research into the process that guards us from mutations that could lead to cancer and other diseases.
Teams led by Drs. Daniel Durocher and Frank Sicheri, of the LTRI, and John Rubinstein, of SickKids, used Electron Cryomicroscopy (Cryo-EM) to see how a protein called 53BP1, a key component of DNA repair machinery, recognizes histones, little balls of proteins that organize our DNA. Cryo-EM is gaining popularity as a high-resolution imaging method of choice, allowing scientists to see atoms that build our body’s molecules.
“This work helps us answer how cells recognize and respond to the alarm caused by damage to our DNA. We were able to see, with this powerful microscope, that 53BP1 decodes the chemical signal that is triggered by DNA damage,” says Durocher, who is also a Professor in the University of Toronto’s Department of Molecular Genetics and Canada Research Chair in Molecular Genetics of the DNA Damage Response.
DNA damage is inevitable – whether it’s harmful products of our own metabolism or environmental factors, all these can cause unwanted genetic changes. Luckily, the cells have evolved efficient repair mechanisms. In one of them, 53BP1 quickly binds to the site of damage and recruits other components of the repair machinery.
Inside the cells, the DNA is tightly wound up around the histones. Should a piece of DNA get broken by, say radiation, the nearby histones become swiftly adorned with chemical tags, which signal to 53BP1 that help is needed. But how 53BP1, or other cellular machineries, read this “histone code” has been a long-standing question in biology.
“We knew that certain modifications happen near the damaged DNA and we knew that 53BP1 was recruited to the sites of damage, but we really had no idea how 53BP1 recognized those modifications,” says Durocher.
To answer this question, Dr. Marcus Wilson, a postdoctoral researcher in Durocher’s team, teamed up with Dr. Samir Benlekbir from Rubinstein’s group that is among the world-leading in Cryo-EM. Thanks to recent technological improvements, Cryo-EM now stands almost neck-to-neck with the gold standard, but technically challenging, method of X-ray crystallography.
Unlike X crystallography, Cryo-EM requires a small amount of material that is relatively easy to prepare. It works by snap-freezing a small volume of liquid containing the sample so that molecular complexes, including the whole lot — the DNA, wound up around the chemically-tagged histones, with 53BP1 bound to them — are randomly arranged in a thin layer of ice. Then, the complexes are irradiated with an electron beam to capture their images from all possible angles. Finally, software sifts through tens of thousands of snapshots to compute a high-resolution 3D model of the entire molecular structure. It worked so well that it caught the researchers by surprise when they realized they were able to see parts of molecules as small as half a nanometer (that’s half a billionth of a metre!) in size.
“Many in the field will be surprised by how high a resolution we got with our particular hardware - near the X crystallography resolution – and at a sufficient resolution to answer many biological questions about how 53BP1 recognizes the damaged site on the DNA,” says Rubinstein, who is also a U of T Professor at the Departments of Biochemistry and Medical Biophysics and Canada Research Chair in Electron Cryomicroscopy.
The long-awaited 3D model not only lets scientists see exactly how 53BP1 is nestled between the DNA and the histones, but it also underscores the importance of the histone code. If even a single tag is missing, or is in a wrong place, 53BP1 will not bind it.
“This is the first time anyone’s seen how a component of the DNA repair machinery reads and interprets the histone code. It’s a really exciting time for EM and it is allowing us to see how the machinery of the cell works with unprecedented clarity,” says Wilson.
This research was primarily supported by operating grants from the Canadian Institutes of Health Research and the Krembil Foundation.
Author: Heidi Singer
A University of Toronto scientist has developed a new method for identifying the raw ingredients necessary to build ‘biologics’, a powerful class of medications that has revolutionized treatment of diseases like rheumatoid arthritis and some cancers.
Biologics are a type of drug that results from the high-tech manipulation of our own proteins, as opposed to more traditional drugs built from synthetic chemicals. Because of their success so far, scientists are racing to create new biologics – and now, a University of Toronto researcher has developed a way to make that process more powerful.
Philip M. Kim, an associate professor in U of T’s Donnelly Centre for Cellular and Biomolecular Research, combined high-tech computer simulation and high-throughput laboratory experiments to create what he hopes will be the most effective way to discover the proteins that are key to new biologics. His research was published online in the journal Science Advances on July 20, 2016.
“A large fraction of new therapeutics these days involve engineered proteins that latch onto a drug target, for instance on a cancer cell,” says Kim, also of the departments of Molecular Genetics and Computer Science. “Finding a protein that effectively binds to a target can feel like looking for a needle in a haystack. Our method should open up new opportunities to find those key proteins – and make a major impact on the development of new biologics.”
Under the traditional approach to developing a biologic, researchers identify a protein of interest and then test billions of variants, either randomly generated or from a natural source, hoping to find an effective binder. But these methods allow very little control over where and how the protein performs this crucial function on its target – a major factor in its effectiveness.
Kim and his team took a different approach. They used a computer to simulate the binding process, and then designed proteins that would work on the target. This type of theoretical approach has been in development for several decades, but is still not effective enough. So Kim combined the best of both methods. Instead of randomly creating massive libraries of variants, as with the traditional approach, he used computer modelling to generate a smaller, but intelligently designed repertoire of variants. Designing each variant allows for the tight control of all its properties, in contrast to conventional approaches.
“We showed that this method gives you binders that are somewhat stronger than what you get with the conventional approach,” says Kim. “The much smaller library also solves many technical problems, and we can screen for new, previously unscreenable, targets. It’s a very exciting time for cancer research, and for biologics.”
For Kim, the next step is to produce proteins that are important to certain types of cancer, but have not been screened before due to the difficulty producing them.
Via Faculty of Medicine News, University of Toronto
When a newt loses a limb due to injury, it simply grows back. Mammals are not as fortunate as evolution has left us without this useful regenerative capacity. One exception however, is the fingertip which regenerates from the distal tip (farthest end of finger) to the nailbed in both mice and humans. How this occurs has been largely unknown.
A new SickKids-led study suggests that nerve-derived cells may play a key role in regulating tissue repair and regeneration in the distal tip and throughout the body.
The research team found that after digit tip injury in adult mice, cells that initially create nerves in the digit tip return to their original regenerative state and secrete growth factors that promote regeneration of the injured fingertip. When this response was prevented, nail and bone regeneration was impaired.
“We have been interested for a long time in how stem cells contribute to the ability of adult tissues to repair themselves, and digit tip regeneration is the ultimate example of repair. In this case, we found that it's not just a single tissue, like skin, repairing itself, but many tissues being repaired in a highly organized fashion,” says Dr. Freda Miller, principal investigator of the study and Senior Scientist in Neurosciences & Mental Health at SickKids.
Miller and her team were surprised to find that nerves contain a population of cells that after injury migrate into the injured tissues and promote tissue repair. She adds that these results demonstrate that this is true of digit tip regeneration, of skin repair, and that it may be important for tissue repair throughout the body too.
This research was supported by the Canadian Institutes of Health Research (CIHR), an Ontario Stem Cell Initiative fellowship, an Ontario Institute of Regenerative Medicine fellowship and SickKids Foundation.
For more information please see the full paper published in the June 30 online edition of Cell Stem Cell.
Published June 30, 2016 by the Hospital for Sick Children
Jun 14, 2016
As the most common and aggressive cancerous brain tumour in adults, glioblastoma is lethal due to its resistance to all currently available treatments. Now, a University of Toronto professor has made a key discovery: glioblastoma seems to like the common neurotransmitter dopamine.
The discovery, by Professor Peter Dirks, of the Division of Neurosurgery and his team at The Hospital for Sick Children, opens new areas of research into treatments for the deadly disease – particularly involving ordinary brain chemicals better known for their role in neurodegenerative conditions like Parkinson’s disease.
Scientists have long understood that when the brain lacks sufficient amounts of dopamine, Parkinson’s disease will occur. In the new study, the team identified chemicals that block dopamine function in glioblastoma tumours in the lab, essentially making glioblastoma stem cells (the cells that drive tumour formation) undergo a rapid neurodegenerative process. But instead of causing Parkinson’s, this process degraded or killed the gliobastoma stem cells. The paper is published in the June 13 online edition of Cancer Cell.
Dirks, a senior scientist at SickKids, spoke to writer Heidi Singer about his discovery.
This is fascinating research. Could it open up other new avenues into the connection between brain chemistry and brain cancer?
Thank you, and, we think this work could possibly open up new avenues. Our study involved an initial screen with many brain neurochemical modifiers. We found that signals involving dopamine, serotonin and other areas of the parasympathetic nervous system also affected glioblastoma cell growth. We are pursuing some of these in further research in the lab.
Why do you think brain chemistry has been an underexplored research area for glioblastoma?
I think we need to look harder at normal brain development and function to get clues about what affects brain tumor cells. These tumors arise from normal cells after all, and despite many genetic mutations, they still (perhaps surprisingly) retain many of the same processes for survival and growth. I believe we need more research that looks at brain tumors through a lens of neurobiology. Not many neurobiologists study brain cancer and vice versa, and this kind of work perhaps falls through a crack as a consequence.
Scientists have noticed that the dopamine-depleted (such as people with Parkinson’s) seem to have fewer glioblastomas. Does it follow that people with higher levels of dopamine could be at greater risk for brain tumors?
We are just scratching the surface of this biology, so the answer is we don’t know, but I doubt that too much dopamine causes a tumor. Our data does not say anything about the emergence of tumors with low or high dopamine — it just says that when you have a glioblastoma, that dopamine signalling plays some role in cell survival. More research is needed!
It’s exciting to think that drugs already on the market could be put to use right away for patients with glioblastomas. How would this work, and how soon can this start?
There are first important safety concerns as many of these agents could affect mood and behaviour, and we don’t know about the long-term use. However, some similar compounds have been tried in people so there is some experience, but we really need more information and further study before they might be used for patients. The goal would be to specifically alter dopamine signalling that is used by the tumor, for example, through the DRD4 receptor, but not to affect other essential dopamine-medicated brain processes. This goal demands more clinically focused research.
We seem to be finding that conditions like schizophrenia, autism, dementia and even ordinary depression have more in common with each other than we ever thought, because they share common pathways. Would you now add brain cancer to that list?
That’s a provocative statement. I think all we can say right now is that brain tumors, like normal brain cells, use some of these pathways also for cell survival.
What inspired your research into glioblastomas?
We are surprised and excited by our findings and the potential clinical application. As a neurosurgeon, I’m continually inspired by my patients. We need to do so much better for them. Hopefully, this recent work opens some new doors and will inspire additional research so we can bring better treatments or treatment combinations to the clinic.
Article courtesy of Faculty of Medicine, University of Toronto
By: Carolyn Morris
Unlike the environmental extremes found in nature, our bodies are fairly stable homes for the trillions of microorganisms we host. Thanks to homeostasis, we keep things like temperature and acidity at regular levels. But we can turn up the heat and run a fever, especially in reaction to an infection. As it turns out, certain opportunistic pathogens — microbes that are normally harmless but can become life-threatening when our bodies are compromised — can thrive in response to this heat shock. In a study published today in Nature Communication, molecular genetics professor Leah Cowen reveals the molecular culprits and the circuitry involved in helping the opportunistic fungus Candida albicans respond to heat. She spoke with Faculty of Medicine writer Carolyn Morris about her findings.
What did you find out about how this fungus responds to heat?
We discovered that simply exposing Candida cells to a 10-minute heat shock, with a sudden upwards shift in temperature, caused the cells to become more destructive in multiple infection models. In response to the heat, Candida activates a transcription factor called Hsf1 — which is a protein that binds to specific sequences in DNA. In this case, it binds to over 100 target genes, controlling their expression and enabling a cellular response to cope with the temperature stress.
What we found was happening is that another protein that normally represses Hsf1 — what we call a “chaperone protein,” because it helps other proteins fold into shape — became so busy refolding proteins that were unraveling due to the increased temperature, that it stopped keeping Hsf1 in check.
In addition, this same chaperone protein was also involved in determining which segments of the DNA would be accessible to the Hsf1 transcription factor. It did this by influencing which areas of DNA are kept folded up tightly in packaging units called nucleosomes, and which areas are left unpacked. This explained why Hsf1 wasn’t binding to all the regions it should recognize.
So not only does this chaperone protein release its hold on Hsf1, it also gives it access to specific sections of DNA, helping to induce the heat shock reaction that makes Candida stronger and more harmful to its host.
Could you liken this to a teenager left home alone without supervision? As if the Hsf1 transcription factor, no longer under the thumb of its chaperone, carried out the cellular equivalent of a raucous house party — a heat shock response?
I’m not sure if the house party analogy works well. To me a teenage house party implies chaos and destruction, when in fact what we found was the opposite. Far from chaotic, this was an extremely orchestrated program required for survival, so protective in nature. I see it more as a carefully crafted scheme or escape plan — one that gets triggered into action when faced with the threat of increased temperature. It’s a great defense mechanism for Candida — but possibly not so great for its host.
What could your findings mean for how we treat pathogens like Candida albicans?
The findings make us revisit longstanding debates about the benefits of fever. Our results suggest that a very short fever might actually help the pathogen turn on programs that make it more dangerous. It may all be a matter of timing, though, considering that other studies we’ve pursued have shown that prolonged elevated temperatures can actually cripple fungal pathogens and make them more sensitive to antifungal drugs. While our studies raise very intriguing ideas about the impact of elevated temperatures on human pathogens, it’s important to recognize that conditions are very different within the human host. We would need detailed clinical studies to gain an appreciation of the actual therapeutic implications.
Are the same players involved in Candida’s response to heat also involved in other infections?
The core cellular regulators that our work focused on are highly conserved in the eukaryotic tree of life, which means they can be found even in species that are distantly related. This suggests that our findings may have broad relevance to fungal pathogens, and perhaps also protozoan parasites, such as those that cause malaria. Notably, fever is a very common response to diverse infections, so there are likely to be complementary mechanisms to respond to temperature fluctuations in bacteria and other microbes.
What do you hope to research next, based on these findings?
Our new work in this area will further probe how cells respond to prolonged elevated temperature. We are taking functional genomic approaches to identify all the genes that are important for cellular responses to high temperature. We’re hoping this will help us identify new strategies to cripple fungal pathogens.
By Jovana Drinjakovic
New tool allows scientists to understand the role of non-coding RNAs
What used to be dismissed by many as “junk DNA” is back with a vengeance as growing data points to the importance of non-coding RNAs (ncRNAs) – genome’s messages that do not code for proteins — in development and disease. But our progress in understanding these molecules has been slow because of the lack of technologies that allow the systematic mapping of their functions.
Now, Professor Benjamin Blencowe’s team at the University of Toronto’s Donnelly Centre, including lead authors Eesha Sharma and Tim Sterne-Weiler, have developed a method, described in May 19 issue of Molecular Cell, that enables scientists to explore in depth what ncRNAs do in human cells. The study is published on the same day with two other papers in Molecular Cell and Cell, respectively, from Dr. Yue Wan’s group at the Genome Institute of Singapore and Dr. Howard Chang’s group at Stanford University in California, who developed similar methods to study RNAs in different organisms.
Of the 3 billion letters in the human genome, only two per cent make up the protein-coding genes. The genes are copied, or transcribed, into messenger RNA (mRNA) molecules, which provide templates for building proteins that do most of the work in the cell. Much of the remaining 98 per cent of the genome was initially considered by some as lacking in functional importance. However, large swaths of the non coding genome – between half and three quarters of it — are also copied into RNA.
What the resulting ncRNAs might do depends on whom you ask. Some researchers believe that most ncRNAs have no function, that they are just a by-product of the genome’s powerful transcription machinery that makes mRNA. However, it is emerging that many ncRNAs have important roles in gene regulation. This view is supported in that some ncRNAs act as carriages for shuttling the mRNAs around the cell, or provide a scaffold for other proteins and RNAs to attach to and do their jobs.
But the majority of available data has trickled in piecemeal or through serendipitous discovery. And with emerging evidence that ncRNAs could drive disease progression, such as cancer metastasis, there was a great need for a technology that would allow a systematic functional analysis of ncRNAs.
“Up until now, with existing methods, you had to know what you are looking for because they all require you to have some information about the RNA of interest. The power of our method is that you don’t need to preselect your candidates, you can see what’s occurring globally in cells, and use that information to look at interesting things we have not seen before and how they are affecting biology,” says Eesha Sharma, a PhD candidate in Blencowe’s group who, along with postdoctoral fellow Tim Sterne-Weiler, co-developed the method.
The new tool, called ‘LIGR-Seq’, captures interactions between different RNA molecules. When two RNA molecules have matching sequences – strings of letters copied from the DNA blueprint – they will stick together like Velcro. The paired RNA structures are then removed from cells and analyzed by state-of-the-art sequencing methods to precisely identify the RNAs that are stuck together.
“Most researchers in the life sciences agree that there’s an urgent need to understand what ncRNAs do. This technology will open the door to developing a new understanding of ncRNA function,” says Blencowe, who is also a professor in the Department of Molecular Genetics.
Not having to rely on pre-existing knowledge is one strength of the method that will boost the discovery of RNA pairs that have never been seen before. The other is that scientists can for the first time look at RNA interactions as they occur in living cells, in all their complexity, unlike in the juices of mashed up cells that they had to rely on before. This is a bit like moving on to explore marine biology from collecting shells on the beach to scuba-diving among the coral reefs where the scope for discovery is so much bigger
ncRNAs come in multiple flavours: there’s rRNA, tRNA, snRNA, snoRNA, piRNA, miRNA, and lncRNA, to name a few, where prefixes reflect the RNA’s place in the cell or some aspect of its function. But the truth is that no one really knows the extent to which these ncRNAs control what goes on in the cell, nor how they do this. The new technology developed by Blencowe’s group has been able to pick up new interactions involving all classes of RNAs and has already revealed some unexpected findings.
The team discovered new roles for small nucleolar RNAs (snoRNAs) that normally guide chemical modifications of other ncRNAs. It turns out that some snoRNAs can also regulate stability of a set of protein-coding mRNAs. In this way, snoRNAs can also directly influence which proteins are made, as well as their abundance, adding a new level of control in cell biology. And this is only the tip of the iceberg as the researchers plan to further develop and apply their technology to investigate the ncRNAs in different settings.
“We would like to understand how ncRNAs function during development. We are particularly interested in their role in the formation of neurons. But we will also use our method to discover and map changes in RNA-RNA interactions in the context of human diseases,” says Blencowe.
Davidson Lab - Study reveals new mechanisms by which viral proteins can turn off the CRISPR adaptive immune system of bacteria
Study reveals new mechanisms by which viral proteins can turn off the CRISPR adaptive immune system of bacteria (Nature 2015, 526, 136-9). The battle for survival between bacteria and the viruses that infect them has led to the evolution of many bacterial systems to defend against these invaders. CRISPR-Cas, one of the most widespread of these systems, is an adaptive system that that specifically targets viral genomes and stores a memory of previous infections. In prior work, a team led by Dr. Alan Davidson and his then graduate student Joe Bondy-Denomy identified the first examples of proteins produced by bacterial viruses that can inhibit CRISPR–Cas systems. In this paper, they elucidated the mechanisms of action of three of these anti-CRISPR proteins, and found that each functions in a distinct manner. This work provides insight into a completely novel group of proteins and increases our understanding of CRISPR-Cas systems, which have recently been developed into powerful tools for human genome editing.
Sidhu Lab - Development of a toolkit of variants of the small protein ubiquitin that modulate function of a core regulator of cellular biology that is misrelated in numerous diseases
Development of a toolkit of variants of the small protein ubiquitin that modulate function of a core regulator of cellular biology that is misrelated in numerous diseases (Molecular Cell 2016, 62:121-36). Team led by Dr. Sachdev Sidhu built upon the powerful high throughput synthetic antibody generation pipeline of the TRAC (Toronto Recombinant Antibody Centre), using phage-displayed libraries of small protein variants to generate biologically active modulators of protein function. Postdoctoral fellow Wei Zhang led the effort to characterize ubiquitin variants targeting HECT E3 ubiquitin ligases, which act as both inhibitors and activators of protein function. They established a general strategy for the highly efficient, systematic development of modulators targeting families of signaling proteins.
Greenblatt Lab - Discovery of a molecular switch that links RNA Polymerase II, a key genome-reading machinery
Discovery of a molecular switch that links RNA Polymerase II, a key genome-reading machinery, with neurodegenerative disorders, shedding light on how some of these devastating diseases may begin (Nature 2016, 529: 48-52). Team led by Dr. Jack Greenblatt found that RNA polymerase II (RNAPII), the key enzyme that puts the RNA together, becomes adorned with chemical tags called methyl groups. In the absence of these tags, RNAPII can’t work with other proteins that help disengage the newly synthesized RNA molecule from the DNA original. This results in the snarling of the DNA and RNA strands, known as R-loops. If left unresolved, R-loops can lead to genome damage. In addition, they can also affect other steps in protein production such as RNA splicing, a process that brings the correct protein-coding parts together in the transcribed RNA. Failure to do so would cause ripples of badly formed proteins that would be damaging to the cell. Greenblatt’s team found that methyl groups on RNAPII help the enzyme recruit a protein called SMN, known to be involved in spinal muscular atrophy, a fatal motor neuron degenerative disease of infancy, and senataxin, which is sometimes mutated in amyotrophic lateral sclerosis, a motor neuron disease that affects speaking, swallowing and eventually breathing. Click here to read more.