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Bacterial enzyme could become a new target for antibiotics

Scientists discover the structure of an enzyme, found in the human gut, that breaks down a component of collagen.

Anne Trafton | MIT News Office
March 17, 2020

MIT and Harvard University chemists have discovered the structure of an unusual bacterial enzyme that can break down an amino acid found in collagen, which is the most abundant protein in the human body.

The enzyme, known as hydroxy-L-proline dehydratase (HypD), has been found in a few hundred species of bacteria that live in the human gut, including Clostridioides difficile. The enzyme performs a novel chemical reaction that dismantles hydroxy-L-proline, the molecule that gives collagen its tough, triple-helix structure.

Now that researchers know the structure of the enzyme, they can try to develop drugs that inhibit it. Such a drug could be useful in treating C. difficile infections, which are resistant to many existing antibiotics.

“This is very exciting because this enzyme doesn’t exist in humans, so it could be a potential target,” says Catherine Drennan, an MIT professor of chemistry and biology and a Howard Hughes Medical Institute Investigator. “If you could potentially inhibit that enzyme, that could be a unique antibiotic.”

Drennan and Emily Balskus, a professor of chemistry and chemical biology at Harvard University, are the senior authors of the study, which appears today in the journal eLife. MIT graduate student Lindsey Backman and former Harvard graduate student Yolanda Huang are the lead authors of the study.

A difficult reaction

The HypD enzyme is part of a large family of proteins called glycyl radical enzymes. These enzymes work in an unusual way, by converting a molecule of glycine, the simplest amino acid, into a radical — a molecule that has one unpaired electron. Because radicals are very unstable and reactive, they can be used as cofactors, which are molecules that help drive a chemical reaction that would otherwise be difficult to perform.

These enzymes work best in environments that don’t have a lot of oxygen, such as the human gut. The Human Microbiome Project, which has sequenced thousands of bacterial genes from species found in the human gut, has yielded several different types of glycyl radical enzymes, including HypD.

In a previous study, Balskus and researchers at the Broad Institute of MIT and Harvard discovered that HypD can break down hydroxy-L-proline into a precursor of proline, one of the essential amino acids, by removing the hydroxy modification as a molecule of water. These bacteria can ultimately use proline to generate ATP, a molecule that cells use to store energy, through a process called amino acid fermentation.

HypD has been found in about 360 species of bacteria that live in the human gut, and in this study, Drennan and her colleagues used X-ray crystallography to analyze the structure of the version of HypD found in C. difficile. In 2011, this species of bacteria was responsible for about half a million infections and 29,000 deaths in the United States.

The researchers were able to determine which region of the protein forms the enzyme’s “active site,” which is where the reaction occurs. Once hydroxy-L-proline binds to the active site, a nearby glycine molecule forms a glycyl radical that can pass that radical onto the hydroxy-L-proline, leading to the elimination of the hydroxy group.

Removing a hydroxy group is usually a difficult reaction that requires a large input of energy.

“By transferring a radical to hydroxy-L-proline, it lowers the energetic barrier and allows for that reaction to occur pretty rapidly,” Backman says. “There’s no other known enzyme that can perform this kind of chemistry.”

New drug target

It appears that once bacteria perform this reaction, they divert proline into their own metabolic pathways to help them grow. Therefore, blocking this enzyme could slow down the bacteria’s growth. This could be an advantage in controlling C. difficile, which often exists in small numbers in the human gut but can cause illness if the population becomes too large. This sometimes occurs after antibiotic treatment that wipes out other species and allows C. difficile to proliferate.

“C. difficile can be in your gut without causing problems — it’s when you have too much of it compared to other bacteria that it becomes more problematic,” Drennan says. “So, the idea is that by targeting this enzyme, you could limit the resources of C. difficile, without necessarily killing it.”

The researchers now hope to begin designing drug candidates that could inhibit HypD, by targeting the elements of the protein structure that appear to be the most important in carrying out its function.

The research was funded by the National Institutes of Health, a National Science Foundation Graduate Research Fellowship, Harvard University, a Packard Fellowship for Science and Engineering, the NSERC Postgraduate Scholarship-Doctoral Program, an Arnold O. Beckman Postdoctoral Fellowship, a Dow Fellowship, and a Gilliam Fellowship from the Howard Hughes Medical Institute.

Cathy Drennan earns Dorothy Crowfoot Hodgkin Award
The Protein Society
March 13, 2020

Protein Society Awards

The nominating process for the 2021 Protein Society Awards is now open. To learn more and submit your nomination for one of our seven awards, click here. Membership is required to submit a nomination, but the nominee does not have to be a member of the Society.

TPS awards recognize excellence across the diverse disciplines that collectively advance our understanding of proteins; their structure, function, design, and application. The Awards honor researchers who have distinguished themselves with significant achievements in protein research and those who have made outstanding contributions in leadership, teaching, and service. TPS members submit nominations, which are awarded by Executive Council, and recipients are honored at the Annual Symposium.

Catherine Drennan,
2020 Dorothy Crowfoot Hodgkin Award Winner

(Massachusetts Institute of Technology)

The 2020 recipient is Professor Catherine Drennan (Massachusetts Institute of Technology). Dr. Drennan  has made enormous contributions  by solving high-resolution structures of proteins and protein complexes that  enhance our understanding of the biology of metalloproteins. Dorothy Crowfoot Hodgkin was famous for using X-ray crystallography to determine the structure of Vitamin B12, and Dr. Drennan has provided monumental insights into the structure and function of proteins that bind to B12.  Dr. Drennan is known for going beyond single proteins and elucidating structures that illuminate entire pathways, capturing multiple snapshots of enzymes as they proceed through their reaction cycles. Among her many notable accomplishments, Dr. Drennan determined the first structure of the cobalamin-dependent ribonucleotide reductase, one of the three enzymes that catalyze the final step in production of deoxyribonucleotides in all organisms. Dr. Drennan’s insights are solidly etched into textbooks and the fabric of our field. Drennan is also an outstanding and widely recognized educator and a tireless advocate for inclusion and equity in science. 

Dorothy Crowfoot Hodgkin Award

Dorothy Crowfoot Hodgkin was a founder of protein crystallography as well as a Nobel laureate. The Dorothy Crowfoot Hodgkin Award, sponsored by Genentech, is granted in recognition of exceptional contributions in protein science which profoundly influence our understanding of biology.
Pioneering researcher Jonathan Weissman joins MIT Biology
Whitehead Institute
March 9, 2020

Whitehead Institute announced today that the globally respected cell biologist Jonathan Weissman has become the Institute’s newest Member and will be the inaugural Landon T. Clay Professor of Biology at Whitehead Institute. Weissman has also been appointed a Professor of Biology at Massachusetts Institute of Technology (MIT). Until joining the Institute, Weissman was Professor and Vice Chair of Cellular and Molecular Pharmacology at University of California, San Francisco (UCSF). He has also been—and continues to be—a Howard Hughes Medical Institute (HHMI) Investigator.

“Jonathan’s extraordinary scientific creativity and productivity, entrepreneurial spirit, and profound expertise will mesh perfectly with Whitehead Institute’s community of innovative, accomplished, and deeply knowledgeable investigators,” says David C. Page, Whitehead Institute Director and Member. “We are thrilled that he will join our quest for new knowledge that ultimately leads to improved human health.”

Weissman is globally renowned for both scientific discovery and building innovative research tools. At Whitehead Institute, he will continue to study the mechanisms used by cells to ensure the correct folding of proteins; develop experimental and analytical tools and approaches to investigate the organization of complex biological systems; and work to develop new applications of the CRISPR-Cas9 gene editing system for biological research and the development of new therapeutics.

“I am excited to be part of the extraordinary communities of Whitehead Institute, MIT, and greater Boston,” says Weissman, who has already built strong research connections in those communities. Those include robust collaborations with MIT’s Aviv Regev and Tyler Jacks; and service on the Science Advisory Board of the Klarman Cell Observatory at Broad Institute. Indeed, Weissman knows Cambridge well, having earned a B.A. in Physics from Harvard University in 1988 and a Ph.D. in Physics from MIT in 1993. At MIT, he conducted research in the lab of former Whitehead Institute Member Peter Kim, where he began studying protein folding. He went on to conduct postdoctoral research at Yale University from 1993 to 1996, working with Arthur Horwich to study the mechanism of GroEL, a molecule key to proper protein folding. Weissman was appointed to the UCSF faculty in 1996 and was named an HHMI Investigator in 2000.

“Jonathan is an outstanding scientist, mentor, and public citizen. He has done transformative work, has developed and applied powerful new technologies, and shared those technologies generously and broadly,” says Alan D. Grossman, Praecis Professor of Biology and Department Head. “Jonathan combines breadth and depth of analyses in ways that very few others can do and we are tremendously pleased that he will be part of the communities at the Whitehead Institute, the Biology Department, and MIT.”

Weissman has published more than 220 peer-reviewed studies, plus numerous research review articles and book chapters; and he has delivered scores of invited lectures and addresses around the world, including the 2019 TY Chen Lecture in Chemical Biology at MIT. A talented educator, Weissman has mentored many leading researchers—notably including Whitehead Fellow Silvi Rouskin and MIT Assistant Professor of biology Gene-wei Li.

Among his many scientific achievements, Weissman developed the ribosome profiling approach that has transformed researchers’ ability to probe the molecular mechanism of translation in vivo; and his lab has subsequently elucidated many fundamental aspects of translation. In addition, Weissman and colleagues—including Stanley Qi, Assistant Professor of Bioengineering and of Chemical and Systems Biology at Stanford University, and Jennifer Doudna, Professor of Biochemistry, Biophysics and Structural Biology at the University of California, Berkeley (UC Berkeley)—developed a CRISPR-associated catalytically inactive dCas9 protein as a general platform for RNA-guided DNA targeting. That work revealed the potential of using “CRISPR interference”—now known as CRISPRi—to precisely regulate gene expression and drive a new type of therapeutic discovery.

Earlier this year, Weissman and Doudna were named as two of the co-leaders of the new Laboratory for Genomic Research (LGR), a $67 million, five-year partnership funded by GlaxoSmithKline to drive development of CRISPR-based therapeutics. Weissman will retain his leadership role in LGR and will maintain close connection with the Innovative Genomics Institute, a joint initiative of UCSF and UC Berkeley that focuses on unraveling the mechanisms underlying CRISPR-based genome editing and applying this technology to improve human health.

In addition, Weissman continues to be a member of the Chan Zuckerberg BioHub’s President’s Advisory Group; to chair the Scientific Advisory Board (SAB) of the Stowers Institute of Medical Research; and to serve on SABs for Amgen and the Helen Hay Whitney Foundation.

An elected member of the National Academy of Sciences (NAS), Weissman received the Protein Society’s Irving Sigal Young Investigator Award in 2004, the Raymond and Beverly Sackler International Prize in 2008, and the NAS Award for Scientific Discovery in 2015. This coming April, Weissman will receive the Genetics Society of America’s Ira Herskowitz Award for outstanding contributions in the field of yeast research in the last 20 years.

Weissman holds five patents (with five more pending) and has been a founder of two biotech companies: Maze Therapeutics and Kendall Square-based KSQ Therapeutics.

Enduring connections

During graduate school, the Schmidts formed lasting ties to the MIT Biology community and one another, which continue to strengthen over time.

Raleigh McElvery
March 9, 2020

Eric and Tracy Schmidt arrived at the MIT Department of Biology in 1990, just two strangers excited to begin their PhDs. Six years later, they left with critical thinking skills that extended beyond the lab bench, and — perhaps more importantly — with each other, marrying in 1999. It’s been over two decades since the Schmidts graduated, but their ties to the department remain strong. Both continue to give back to the department, in order to support the mentorship opportunities and cutting-edge research they experienced as graduate students.

Before coming to MIT, Eric completed his degree in chemistry at the University of Pennsylvania. He aimed to understand life at a molecular level, and determined a PhD in biology would complement his chemistry background. He applied to MIT Biology because it was one of the first life science departments in the nation to embrace this molecular focus. “I was keen to immerse myself in the science and learn alongside the very best, from the very best,” he says. His father had also attended MIT for graduate school, which only made Eric more enthusiastic about joining the Institute.

Tracy (née Tracy Smith) received her bachelor’s degree from Carleton College in Minnesota, majoring in biology. She chose to pursue graduate work at MIT Biology because the department’s energy was, as she puts it, “palpable.” From collecting data in the lab to attending lectures and striking up casual conversations with colleagues, she can still recall the intense intellectual atmosphere. “MIT Biology was, and continues to be, a cutting-edge and collaborative research community,” she says.

Tracy worked in Robert Sauer’s lab studying cooperative DNA binding and transcriptional regulation, while Eric was in Paul Schimmel’s lab investigating transfer RNA synthetases. But they both learned more than just the scientific principles of their respective fields — they gleaned analytical skills, strong work ethics, and the ability to objectively assess the pros and cons of a given situation.

Eric remembers his academic experience as being rigorous, but also collaborative and fun. “The department had a nonhierarchical structure that helped to soften the intensity that is often associated with innovative research,” he says. He recalls playing intramural sports alongside accomplished professors, and discussing current events with senior faculty members over drinks.

Post-MIT, Eric co-founded Cambridge Biological Consultants, and went on to become vice president and research analyst at UBS Securities. Later, he served as the managing director and senior biotechnology analyst at Cowen and Company. In 2018, he joined Allogene Therapeutics as their chief financial officer, furthering their goal of creating off-the-shelf CAR-T therapies to treat cancer.

Tracy went on to complete her postdoc at the University of California, San Francisco under Cori Bargmann PhD ’87, and eventually became the chief editor at Nature Structural Biology (now Nature Structural and Molecular Biology). Since 2001, she’s been working as a freelance scientific writer and editor, and she is also an aspiring children’s book author.

“Both my graduate research at MIT and my work as a scientific editor and writer helped to hone my critical thinking skills, as well as my writing abilities,” she says. “These experiences helped me to develop patience in achieving long-term goals.”

Today, the Schmidts remain active members of the MIT Biology community from their home in New York. As part of the Visiting Committee, Eric returns to campus at least every other year to discuss important trends, provide an industry perspective, and help guide the trajectory of the department.

Both Eric and Tracy continue to assist the department, and serve as a resource for Department Head Alan Grossman. Since graduating, they have supported MIT Biology philanthropically on an annual basis, and will be presented with the 77 Society Medallion later this year for their leadership and generosity.

“Having benefited so much from MIT Biology, we feel fortunate to be able to give something back,” Eric says, and recommends that other alumni get involved with the department. “If you do become involved, I think you’ll find that while much has changed, the department’s goals and values have not, and it is easy to reconnect no matter how long you have been away.”

Their time at MIT allowed Eric and Tracy to form enduring ties to the community — and each other — which continue to strengthen to this day. “MIT has been largely responsible for our family,” Eric says. “We now have three wonderful children, who may or may not pursue a degree in biology.” Tracy agrees, “We have many friends that we met at MIT, and we will always have a bond through shared MIT experiences.”

Chimeras offer a new way to study childhood cancer in mice
Eva Frederick | Whitehead Institute
March 5, 2020

In a new paper published March 5 in the journal Cell Stem Cell, researchers in Whitehead Institute Member Rudolf Jaenisch’s lab introduce a new way to model human neuroblastoma tumors in mice using chimeras — in this case, mice that have been modified to have human cells in parts of their nervous systems. “This may serve as a unique model that you can use to study the dynamic of immune cells within human tumors,” says Malkiel Cohen, a postdoc in Jaenisch’s lab and the first author of the paper.

Neuroblastoma is a rare and unpredictable form of childhood cancer that affects around 800 young children in the US each year. Neuroblastoma tumors often occur in parts of the sympathetic nervous system, which includes the nerves that run parallel to the spinal cord and the adrenal medulla, part of the glands that produce hormones such as adrenaline. Neuroblastoma is notoriously hard to study primarily because of its disparate behavior: the tumors often shrink spontaneously in infants, while in toddlers they are highly aggressive and often fatal. “The seeds for the cancer are sown during fetal life,” says Rani George, MD, PhD, an associate professor of pediatrics at Harvard Medical School and a neuroblastoma researcher and physician at Dana-Farber Cancer Institute and Boston Children’s Hospital, and a co-senior author on the paper. “For obvious reasons, you can’t really study the development of these tumors in humans.”

Until now, researchers didn’t have many realistic ways to study these tumors in animal models, either. They could create transgenic mice with cancer-causing genes, but the resulting tumors were mouse tumors, not human ones, and had some key differences. Another method involved taking human tumor cells and implanting them in a mouse — a process called xenotransplantation — but that only worked in mice with compromised immune systems, and didn’t allow researchers to study how the tumors formed in the first place or how they interacted with a fully functioning immune system. “This is where we think the new model is a perfect fit,” said Stefani Spranger, PhD, an assistant professor of Biology at the Massachusetts Institute of Technology (MIT) and the Koch Institute for Integrative Cancer Research at MIT and a co-senior author on the paper.

Human-mouse chimeras have been used in the past to study Alzheimer’s disease and brain development. Jaenisch, who is also a professor of biology at MIT, and his lab had been working for years to create chimeric mice with human cells in the neural crest — the group of developing cells that go on to form parts of the sympathetic nervous system — and published their findings in 2016. “In this study, we hoped to use these mice with human neural crest cells to study how neuroblastoma tumors form and respond to immune system attacks,” Jaenisch says.

To create these chimeric mice, Cohen and coauthors at MIT’s Koch Institute and the Dana-Farber Cancer Institute first engineered human pluripotent stem cells to express two genes known to be abnormal in neuroblastoma, MYCN and mutated ALK, and modified them so they became neural crest cells, from which human neuroblastomas are derived. The genes could be turned on and off with the addition of doxycycline, an antibiotic. They also inserted the gene for eGFP, a brightly glowing fluorescent protein originally isolated from jellyfish. This would allow the team to tell whether the cells were spreading correctly through the bodies of the mice, and would cause any tumors originating from these human cells to be luminous under fluorescent light.

The researchers injected mouse embryos with these cells, and watched over the course of embryonic development as the cells proliferated and human tissues crept into the developing peripheral nervous systems of the tiny mice. To activate the two cancer-causing genes, researchers spiked the pregnant mice’ water with doxycycline, and over the next few days in utero — and in the weeks and months after the pups were born — the researchers inspected the chimeras to see whether tumors would appear.

Over the course of the next 15 months, 14% of the mice developed tumors — 29 mice out of 198 total. The tumors mostly appeared in the space behind the abdominal cavity close to the nerves along the spinal cord, although one mouse developed a tumor in its adrenal gland. Both locations are common places for human children to develop neuroblastoma. The researchers took samples of the tumors and found that they contained the glowing protein eGFP, which confirmed that they were of human origin.

When the team examined the growth patterns of the cancerous cells, they found that the tumors were remarkably similar to human neuroblastomas: they contained cell markers typical of human tumors, and some grew in characteristic rosette shapes — features that did not often appear in tumors implanted in immunocompromised mice through xenotransplantation.

Having successfully induced neuroblastoma tumors in the chimeric mice, the researchers took the opportunity to examine the communication between immune cells and tumors — and specifically, how the tumors evaded destruction by anti-cancer immune cells called T cells. One factor that makes human neuroblastomas and many other cancers dangerous is their sophisticated strategy for avoiding being destroyed by T cells. “The cancer tricks the immune system,” Cohen says.  By activating chemical signals that exhaust the T cells, the tumors effectively weaken their attack. The tumors in the chimeric mice, Cohen found, use a similar method to human neuroblastomas to evade immune responses.

Cohen and others plan to test the new system’s potential for modeling other cancers such as melanoma, and to use it to investigate potential treatments for neuroblastoma patients. “The obvious next step is to study how treatment of these tumors will allow these chimeric mice to be cured,” he says. “This is a model that will allow us to approach not only how to get rid of the tumor, but also to fix the immune system and recover those exhausted T cells, allowing them to fight back and deplete the tumor.”

This research was funded by the National Institutes of Health, as well as grants from the Emerald Foundation, the LEO Foundation, the Melanoma Research Foundation, and the St. Baldrick’s Foundation.

Citation: Cohen, M., et al. Formation of Human Neuroblastoma in Mouse-Human Neural Crest Chimeras. Cell Stem Cell. March 5, 2020. DOI: https://doi.org/10.1016/j.stem.2020.02.001

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Written by Eva Frederick

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QS World University Rankings rates MIT No. 1 in 12 subjects for 2020

Institute ranks second in five subject areas.

MIT News Office
March 4, 2020

MIT has been honored with 12 No. 1 subject rankings in the QS World University Rankings for 2020.

The Institute received a No. 1 ranking in the following QS subject areas: Architecture/Built Environment; Chemistry; Computer Science and Information Systems; Chemical Engineering; Civil and Structural Engineering; Electrical and Electronic Engineering; Mechanical, Aeronautical and Manufacturing Engineering; Linguistics; Materials Science; Mathematics; Physics and Astronomy; and Statistics and Operational Research.

MIT also placed second in five subject areas: Accounting and Finance; Biological Sciences; Earth and Marine Sciences; Economics and Econometrics; and Environmental Sciences.

Quacquarelli Symonds Limited subject rankings, published annually, are designed to help prospective students find the leading schools in their field of interest. Rankings are based on research quality and accomplishments, academic reputation, and graduate employment.

MIT has been ranked as the No. 1 university in the world by QS World University Rankings for eight straight years.

Exploring How Cells Repair and Tolerate DNA Damage
National Institute of Environmental Health Sciences
March 2, 2020

Graham Walker, Ph.D., studies the processes cells use to repair and tolerate DNA damage from environmental pollutants. For more than 40 years, he has worked to understand how cells respond to DNA damage, and how these processes can introduce mutations that lead to cancer and other human diseases.

His current NIEHS-funded work focuses on translesion synthesis (TLS). This damage tolerance process allows specialized enzymes that copy DNA, called TLS DNA polymerases, to replicate past lesions in damaged DNA. The process can help cells tolerate environmental DNA damage, but because TLS polymerases frequently insert the wrong DNA base, they can also lead to DNA mutations.

“The TLS process is critically important to human health because it helps cells survive DNA damage, but it can come at a cost,” said Walker. “It isn’t the kind of repair system you would think we would want because it makes a lot of mistakes. However, as we drill into these details, we are finding that there is so much more to be learned than just the strict biochemistry.”

In 2017, Walker was one of eight environmental health scientists to receive an inaugural Revolutionizing Innovative, Visionary Environmental Health Research (RIVER) Outstanding Investigator Award from NIEHS. The grant, which funds researchers rather than specific projects, provides Walker with flexibility to explore novel directions in his research.

From the Ames Test to TLS

Walker was drawn into the world of DNA repair and mutagenesis as a postdoctoral fellow at the University of California, Berkeley, under the guidance of Bruce Ames, Ph.D. Ames’ group created the Ames test, still used today, to determine whether a given chemical is likely to cause cancer. The Ames test uses bacterial strains that include a derivative of a naturally occurring drug-resistant plasmid, a small circular DNA molecule, known as pKM101. This molecule significantly increases the mutation rate of bacterial genes in response to chemical exposures, playing an important role in this quick and convenient test to estimate carcinogenic potential.

“I decided there must be something really interesting on that plasmid because it led to much higher mutation rates in bacteria for the same amount of damage,” said Walker.

After arriving at the Massachusetts Institute of Technology, his current employer, Walker continued to study the mechanisms behind these mutations.

Walker and his research team discovered the specific genes of pKM101 that are needed for it to produce more mutations. They showed that these genes are orthologs, or genes that evolved from a common ancestral gene, in the Escherichia coli (E. coli) chromosome that are required for the bacteria to mutate in response to DNA damage. This work helped lay the groundwork for the discovery of TLS DNA polymerases and how they are controlled.

“When we first sequenced these genes, nothing like them had been previously reported, but subsequently more and more related genes were discovered in all domains of life,” said Walker. “After decades of work by many labs, we now know that these are all TLS DNA polymerases and that the pKM101 plasmid encodes a polymerase that is responsible for the increased mutations.”

Using Bacteria to Understand DNA Damage

Walker’s prior research on the mutagenesis-enhancing function of pKM101 also led him to analyze E. coli’s SOS system, a set of biological responses that are activated to rescue cells from severe DNA damage. Walker and his team identified genes turned on by DNA damage that are regulated as part of E. coli’s SOS response. Many of the genes encode functions involved in DNA repair or mutagenesis. This work on the SOS response of E. coli was the first to directly demonstrate, in any organism, that DNA damage from environmental sources can change gene expression.

By further exploring TLS DNA polymerases in E. coli, he also identified the biological role of one of the most conserved DNA-damage response enzymes, DinB, which encodes a TLS DNA polymerase, and reported that the gene is required for resistance to some DNA-damaging agents. His work on DinB also suggested an additional mechanism by which antibiotics can become toxic to bacterial cells.

Blocking TLS in Cancer

“While a postdoc in the mid 1970’s with Bruce Ames, my ambitious hope was that by studying pKM101, I would learn something about the fundamental mechanism of how mutations arose in bacteria and humans, and might even learn how to control it,” said Walker. “That is now happening with my current, NIEHS-funded work.”

Some tumors can withstand damage from chemotherapy drugs by relying on TLS, which allows them to survive by replicating past damaged DNA caused by the drugs. In eukaryotes, including humans, mutagenic TLS is carried by two TLS DNA polymerases known as Rev1 and Pol zeta.

In addition to his innovative research, Walker is devoted to improving education and helping undergraduate students. In 2002, Walker became a Howard Hughes Medical Institute Professor and used his funding to establish a science education group modeled on his laboratory research group.

“I feel that training the next generations of scientists is as important as the science itself, and I have been incredibly lucky to have a spectacular set of grad students and post docs work with me over the years,” said Walker. “I have tried to focus as much on training, through teaching and mentoring, as on advancing the science.”

“Not only are these TLS polymerases responsible for introducing a lot of mutations that cause cancer, they also help cancer cells survive in the face of chemotherapy drugs that introduce DNA damage that would otherwise kill them,” said Walker.

Recently, Walker and his colleagues discovered that a small molecule and compound known as JH-RE-06 can block the Rev1-Pol zeta mutagenic TLS pathway by interfering with the ability of the Rev1 domain to recruit Pol zeta. The researchers tested the molecule in human cancer cell lines and showed that it enhanced the ability of several different types of chemotherapy to kill cancer cells, while also suppressing their ability to mutate in the presence of DNA-damaging drugs. In a mouse model of human melanoma, they found that not only did the tumors stop growing in mice treated with a combination of the chemotherapy drug cisplatin and JH-RE-06, those mice also survived longer.

“I am able to take more chances and try more high-risk experiments with the RIVER award,” said Walker. “The flexibility and extra resources are now allowing me to identify TLS inhibitors, which are offering startlingly unexpected mechanistic insights and also show potential to improve chemotherapy.”

A force for health equity

Through on-site projects in developing countries and internships in the business world, Kendyll Hicks explores the political and economic drivers of global health.

Becky Ham | MIT News Office
March 1, 2020

After spending three weeks in Kenya working on water issues with Maasai women, Kendyll Hicks was ready to declare it her favorite among the international projects she’s participated in through MIT.

As a volunteer with the nonprofit Mama Maji, Hicks spoke about clean water, menstrual hygiene, and reproductive health with local women, sharing information that would enable them to become community leaders. “In rural Kenya, women are disproportionately affected by water issues,” she explains. “This is one way to give them a voice in societies that traditionally will silence them.”

The team also planned to build a rainwater harvesting tank, but climate change has transformed Kenya’s dry season into a rainy one, and it was too wet to break ground for the project. During her stay, Hicks lived in the home of the first female chief of the Masaai, Beatrice Kosiom, whom Hicks describes as “simultaneously a political animal and the most down-to-earth-person.” It was this close contact with the community that made the project especially fulfilling.

During MIT’s Independent Activities Period, Hicks also has traveled to South Africa to learn more about the cultural and biological determinants of that country’s HIV/AIDS epidemic, and to Colombia to lead an entrepreneurial initiative among small-scale coffee farmers. Hicks joined the Kenya trip after taking an MIT D-Lab class on water, sanitation, and hygiene. Each experience has been successively more hands-on, she says.

“I’ve been drawn to these experiences mainly because I love school, and I love the classroom experience,” Hicks says. “But it just can’t compare to living with people and understanding their way of life and the issues they face every day.”

Hicks, a senior majoring in computer science and molecular biology, says she has shifted her focus during her time at MIT from more incremental technical discoveries to addressing larger forces that affect how those discoveries contribute — or fail to contribute — to global health.

Her love of biology began with animals and zoology, later expanding into an interest in medicine. “Humans are these amazing machines that have been crafted by nature and evolution, and we have all these intricacies and mechanisms that I knew I wanted to study further,” Hicks says.

At the same time, she says, “I’ve always been interested in health care and medicine, and the main impetus behind that is the fact that when someone you love is sick, or if you’re sick, you’ll do whatever you can.”

As a first-year student she worked in the Lippard Lab at MIT, helping to synthesize and test anticancer compounds, but she soon decided that lab work wasn’t the right path for her. “I made the realization that health care and medicine are extremely political,” she recalls. “Health policy, health economics, law — those can be the drivers of real large-scale change.”

To learn more about those drivers, Hicks has worked two summers at the management consulting firm McKinsey and Company, and will take a full-time position with the company after graduation.

“As someone immersed in the world of science and math and tech, I had this lingering insecurity that I didn’t know that much about this entirely different but super-important area,” she says. “I thought it would be important to understand what motivates business and the private sector, since that can have a huge effect on health care and helping communities that are often disenfranchised.”

Hicks wants to steer her work at McKinsey toward their health care and hospital sector, as well as their growing global health sector. Over the long term, she is also interested in continuing fieldwork that involves science, poverty eradication, and international development.

“Being at MIT, it’s like this hub of tech, trying to venture further into novel breakthroughs and innovations, and I think it’s amazing,” Hicks says. “But as I have started to garner more of an interest in politics and economics and the highly socialized aspects of science, I would say it’s important to take a pause before venturing further and deeper into that realm, to make sure that you truly understand the downstream effects of what you are developing.”

“Those effects can be negative,” she adds, “and they oftentimes impact communities that already are systematically and institutionally oppressed.”

Hicks joined MIT’s Black Students Union as a first-year student and now serves as the BSU Social and Cultural Co-Chair. In the role, she is responsible for planning the annual Ebony Affair fly-in program, which brings more than 30 black high school students to campus each year to participate in workshops, tour labs, and join a gala celebration with BSU students, faculty, and staff. “We’re doing our best as a community to convince young bright black minds to come to a place like MIT,” she says.

It worked for Hicks: She participated in Ebony Affair as a high schooler, and the experience cemented her decision to attend. “When I saw everyone showing out and having such pride in being black and being at MIT, I was like, ‘OK, I want to be a part of that,’” she recalls.

Last year, Hicks planned BSU’s first Black Homecoming event, a barbecue that brought together current and former black MIT students — some who attended the school 50 years ago. The event was a celebration of support and a way to strengthen the BSU network. “You have to do what you can to cultivate communities wherever you are, and that’s what I’ve tried to do here at MIT,” she says.

Hicks also served as the Black Women’s Alliance alumni relations chair and GlobeMed’s campaigns co-director, and was on the Undergraduate Association Diversity and Inclusion Committee. She has discovered a love of event organizing and leadership at MIT, although it has been a change of pace from her former shy, “hyper-bookworm” self, she says.

“I have realized that in my career that I really want to do a lot of good and affect a lot of change in people’s lives, and in order to do that, you kind of have to be this way.”

Unusual labmates: Biology all-stars
Greta Friar | Whitehead Institute
February 25, 2020

Meet the stars

Zak Swartz, a postdoctoral researcher in Whitehead Institute Member Iain Cheeseman’s lab, gets an unusual delivery a few times a year. It comes in a cardboard box a few feet in length on each side with a “perishable” label on top. When the most recent box arrived, Swartz took it to a small, chilly room across from Cheeseman’s lab. He cut and tore his way through several layers of packaging, insulation, and cold packs to get to his prize: a bunch of plastic bags half-filled with water, the sort of thing that might contain a goldfish at a county fair. Instead of goldfish, these makeshift aquaria each contained two or three bat stars (Patiria miniata), a hardy species of starfish seemingly unphased by their transcontinental trip in a cardboard box.

Bat stars are so named because the thick webbing between their arms—of which they typically have five, though they can have up to nine—gives their short limbs a bat-wing-like appearance. The stars are most often some shade of red or orange, but come in a variety of colors and patterns. Each star that Swartz pulled out of the box had a unique design coating its body.

Swartz untied the plastic bags and took the bat stars out one by one. He gently dropped each star into an aquarium in the corner of the room, where it would sink slowly to the bottom, then crawl to the sides and inch its way up, clinging to the glass. Although their movements are barely perceptible to a human watching, within minutes bat stars coated the walls of the aquarium, the tiny tube feet on the underside of each arm sticking them firmly in place.

As the first round of bat stars settled onto their chosen perches, Swartz returned to the box. He took another bat star out of its bag and held it in his hand for a moment.

“This one’s heavy,” he said, with satisfaction; heavy stars are more likely to be full of eggs, and that’s what Swartz is interested in. He’s researching how cells, such as immature egg cells, that remain dormant or non-dividing for a long time retain their ability to divide. The proteins necessary for cell division degrade over time, like parts of a machine rusting and breaking down, and yet many cells remain able to divide long after their unused cellular machinery should have become useless. In the case of humans, precursor egg cells can spring back into action after decades of dormancy in the ovaries, and can go on to perform that most impressive feat of cell division: the creation of a whole new organism from one cell. But human eggs are not the most accessible or readily available research material, and so Swartz has turned to the bat stars, an excellent source of reproductive cells, to help answer his questions.

Fertile ground for discovery

Bats stars reproduce by spawning. The females release millions of eggs into the ocean through pores in between their arms, while at the same time males release clouds of sperm. The reproductive characteristics of bat stars make them ideal research animals for Swartz. They have a long breeding season during which they can ovulate, they produce millions of eggs at a time, and they release these eggs out into their environment, where they develop externally.

In the lab, Swartz must extract the immature egg cells before they are released, so he can study the processes that take place in the cells during their development. This is much easier than extracting cells from mammalian ovaries; all it requires is a minimally invasive procedure from which the starfish quickly recover. Once Swartz has extracted the eggs in their “hibernating” pre-spawn state, he can control and observe all of the steps of their development, from their re-activation through to fertilization and beyond.

In the wild, fertilized bat star eggs develop into embryos, which quickly become tiny, transparent larvae that swim freely and live as plankton. These larvae have strong regenerative capabilities: if a bat star larva is cut in half, each half can regrow the missing parts of its body. Some species of starfish maintain robust regenerative capacity into their adult stage.
Unlike the adult stage of the bat star, the larvae have bilateral symmetry, with mirroring left and right sides, just like humans. Bat stars develop through several stages as bilateral larva, becoming a bipinnaria and then a brachiolaria. These stages are transparent, their insides easily visible—a good feature for research subjects. Only when the stars metamorphose into their juvenile and then adult forms do they assume their familiar, five-point radially symmetric shape.
Bat stars share a common ancestor with mammals, and bat star and human embryonic development are similar enough for the former to be a useful model for the latter in research like Swartz’s. In fact, bat stars belong to the phylum Echinodermata, a group of animals also including sea urchins, sea cucumbers, sand dollars and others, that have historically proved to be important tools for research into reproduction. The first observation of sperm fertilizing an egg occurred in transparent sea urchin eggs, which helped solve the mystery of how each sex’s gametes contribute to sexual reproduction.

One reason Swartz has chosen bat stars over other starfish species is because of their hardiness. Bat stars fare well in a laboratory aquarium—they even do well being shipped in a cardboard box—whereas other species that Swartz considered using are less adaptable to these conditions. So, while it may have been more convenient to use a species from local Atlantic waters, in the interest of maintaining a healthy lab population of specimens, Swartz has had to procure his research subjects long distance. The geographical range of bat stars is the stretch of Pacific Ocean along the coast of North America.

How are the animals getting from the Pacific coast to Whitehead Institute? They are collected by a contact Swartz made several years ago in California.

Scuba diving for science

Josh Ross runs a research specimen procurement company based in San Pedro, California called South Coast Bio-Marine. Ross has been collecting starfish for Swartz since 2015, and he also collects a variety of marine animals, from sea urchins to limpets to nudibranchs, for researchers at other institutions. Specimens from South Coast Bio-Marine have been used in research on, among other topics, fertilization, memory formation, sleep, and shape changes in oocytes.

Most mornings, Ross and his employees load up a boat with scuba gear and equipment and head to their chosen dive spots, where they collect specimens for the first half of the day.

“I love the fact that I get to dive and work in the ocean every day. When Monday comes, all the weekend boaters and fishermen go back to their jobs, and we have the ocean almost all to ourselves. We are out in the wilderness with truly wild ocean creatures,” Ross says—though sometimes those wild creatures can interfere with their collection plans.

The team then take what they have collected back to Ross’ lab, where the specimens are kept in chilled seawater tanks for a few days to acclimate before Ross ships them to researchers. Ross takes care to harvest specimens with sustainability in mind. When finding starfish for Swartz, he will only take animals that have had time to grow large and spawn several times in their native habitat before being collected.

Ross searches for bat stars at dive sites in 55- to 70-foot deep water, either out in the open, on rocky shelf reefs, or on the sand near the reefs. Bat stars’ habitat ranges from the low intertidal zone, the part of the seashore that’s covered with water except at low tide, into the mild depths of the subtidal zone. They live among kelp and surf grass forest, and use the numerous tube feet lining the underside of each arm to crawl across the sandy ocean floor and cling to rocks. Instead of blood, starfish pump sea water through a water vascular system to circulate nutrients through their bodies and control their limbs. They pump water into and out of their tube feet to make them extend and contract, allowing the stars to move. The tube feet can also release glue-like chemicals that help the stars adhere to rocks even in the strong currents of the ocean—or cling to glass walls in aquariums.

Bat stars are voracious eaters—scavengers as well as predators—and Ross often finds them in the middle of a meal. Bat stars eat by extending their stomachs out of their bodies to dissolve their prey in digestive juices, then drink it up. This system allows them to eat larger prey than their small mouths would otherwise allow. A second stomach that remains inside the star further digests the food.

Ross tries to select starfish for Swartz that feel “ripe,” meaning they are ready or nearly ready to spawn. Indicators that Ross uses include ripe stars having larger “shoulders” and being puffier than unripe stars. After Ross has collected enough bat stars, he ships them from California to Cambridge, Massachusetts, where their role in Swartz’ research begins. Swartz is interested in female specimens, but there is no good way to identify a starfish’s sex on sight, so the bat stars that Ross sends to Swartz tend to be fifty-fifty female and male. Swartz must examine a small biopsy of the gonad to find out which of them contain oocytes, the immediate precursor cells to fertilizable eggs. Once identified, the animals are separated into two aquariums by sex, ready to provide oocytes for experiments. Swartz feeds the starfish a steady diet primarily consisting of raw, peeled shrimp, which keeps them developing new oocytes. When the starfish have completed their time in the lab, Swartz tries to donate them to local aquariums.

Eggs with answers: What we’ve learned from bat stars

Swartz is using the bat stars to investigate how cells divide—specifically, how cells retain the ability to divide after long periods without doing so, and how cell division processes are adapted to the context of animal reproduction and development. Whitehead Member Iain Cheeseman’s lab, where Swartz is a postdoc, investigates the cellular machinery required for cell division. In particular, Cheeseman’s team studies the kinetochore, a complex of proteins involved in orchestrating the precise segregation of chromosomes during cell division, and the centromere, the region in the middle of the chromosome where the kinetochore assembles. The centromere is not defined by its DNA sequence, but by proteins that attach there and signal the kinetochore to assemble at that location. One of the necessary proteins that marks the centromere is called CENP-A. Without CENP-A, the centromere won’t function properly, so chromosomes won’t be correctly distributed into the two new cells created during cell division. However, as with other proteins, there was an open question whether CENP-A degrades over time. Once it is lost at the centromere the cell cannot get it back, and loses the ability to divide. This fact caused Cheeseman and Swartz to wonder how cells that spend long periods of time without dividing can start up again. What sort of maintenance do cells need to do to keep their cell division machinery operational?

Eggs and oocytes, the precursor cells that will develop into eggs, are a great test case because they remain non-dividing for a very long time. Swartz harvested the bat stars’ oocytes and used a fluorescent tag to track the quantity of CENP-A inside of the cells as they progressed through their cell cycle stages. To get a close look at what happens to the CENP-A in oocytes during their dormancy, Swartz maintained the cells in a state of arrested development in petri dishes by putting them in a mixture he calls “starfish juice,” a blend of culture fluids, antibiotics, and some of the bat stars’ own natural fluids.

With the help of the fecund bat stars, Swartz and Cheeseman found the answer to their questions about CENP-A. In research published in Developmental Cell in 2019 [4], the scientists discovered that cells slowly replace their CENP-A over time, swapping out the old protein at risk of breaking down with new functional protein. This finding upended the previous understanding of CENP-A as a static protein that was placed on the centromere once and then remained as long as it could. The researchers also tested a human cell line that can enter dormancy and divide later, and found that, like the sea star oocytes, those cells gradually exchanged CENP-A. In contrast, the researchers discovered that cell types that never need to divide again, like muscle cells or other specialized cells, let most of their CENP-A degrade and so permanently lose the capacity to divide. This finding means that the presence of CENP-A may be a good indicator for use in determining whether any given cell retains the ability to divide in the future. The question of a specialized, or terminally differentiated, cell’s potential for renewed cell division is of great interest in regenerative medicine research. Indeed, this work sparked collaboration between Cheeseman, Swartz and Whitehead Institute Fellow Kristin Knouse, who studies regeneration in mouse and human cells.

The findings could also explain why tissues like muscle rarely develop cancers; the cells cannot replicate and so cannot grow tumors. Furthermore, Swartz thinks that their findings could prove valuable for assisted fertility research.

“Understanding the natural biology that keeps eggs in good shape, able to resume and finish their development after long dormancies, could provide insight into what goes wrong when eggs do not remain viable,” Swartz says.

The possibilities for future research spawning from Swartz’ work are many. The advances that may come, whether in regenerative medicine, assisted fertility, or elsewhere, will all be owed in part to a group of bat stars that travelled across a continent, from ocean to ocean, in a chilled cardboard box to help unravel the mysteries of cell division.

Researchers discover an RNA-related function for a DNA repair enzyme
Raleigh McElvery
February 26, 2020

After decades of speculation, researchers have demonstrated that a classical DNA repair enzyme also binds to RNA, affecting blood cell development.

The DNA-dependent protein kinase, otherwise known as DNA-PK, is one of the most important enzymes that binds DNA and repairs double-stranded breaks. This mode of repair is essential for generating receptors that help the immune system fight off intruders. But DNA-PK doesn’t just bind DNA; it also binds RNA. Although researchers have known this for decades, they didn’t fully understand what kinds of RNAs DNA-PK bound in mammalian cells, or the physiological consequences of this binding.

In a new study published on February 26 in Nature, researchers from MIT and Columbia University have uncovered a mechanism whereby DNA-PK binds to the RNA involved in ribosome assembly. Ribosomes — the cell’s protein synthesis machinery — ensure that stem cells give rise to enough red blood cells. The researchers found that mutating DNA-PK prevents the ribosomes from being built properly, which prevents blood cells from doing their job and leads to blood disorders.

“This is the first biochemical evidence of DNA-PK assembly and activation by RNA inside cells,” says Eliezer Calo, a co-senior author and assistant professor in MIT’s Department of Biology. “We’re still trying to determine the mechanisms that regulate protein synthesis in stem cells, and this study reveals one of them.”

Co-senior author, Shan Zha from Columbia University, had previously studied DNA-PK’s role in DNA repair by generating a mouse model that carried enzymatically-dead versions of DNA-PK. While using this model to investigate tumorigenesis, Zha’s lab found these mutant mice developed a form of blood cancer known as myeloid disease. At the same time, another research group showed that mutations in DNA-PK also led to anemia, which occurs when the body does not have enough healthy red blood cells

Neither myeloid disease nor anemia could be easily explained by DNA repair defects alone. However, the two blood disorders did share some similarities to diseases caused by ribosome defects. Because DNA-PK resides in the same organelle where ribosomes are made, the Zha and Calo labs began to wonder whether DNA-PK could bind to the RNA there and control ribosome biogenesis.

In this new study, the Zha lab found that DNA-PK mutations impaired protein translation in red blood cell progenitors, which might contribute to anemia. In parallel, the Calo lab was investigating ribosomal RNA processing and was surprised to find that DNA-PK seemed to be implicated in ribosome assembly. The Calo lab then mapped all the RNAs in cells that bind DNA-PK. The enzyme unexpectedly attached to U3, a small RNA that helps assemble one of the subunits comprising the ribosome. Once it binds U3, DNA-PK can transfer a phosphate group to several specific sites on one of its own subunits. If DNA-PK is defective and cannot transfer the phosphate group, protein synthesis in blood stem cells is impaired, eventually causing anemia.

DNA-PK is essential for cellular viability in nearly all human cell lines, including cancer cell lines, while many other proteins involved in same DNA repair pathway are dispensable. Several studies, including one published by the Zha lab, showed that DNA-PK protein levels are 50-fold higher in common human cell lines than in rodent cell lines. The researchers do not yet know why the enzyme is so critical, but they suspect it might have to do with its ability to bind RNA. “We are interested in exploring whether this new role for DNA-PK could provide clues to this puzzle,” Zha says.

Calo says their findings could also have important implications for cancer treatment, because DNA-PK has emerged as a promising target for cancer therapy. Drugs that inhibit DNA-PK could prevent cancer cells from repairing their DNA and replicating successfully, but he warns these same remedies could also impact stem cell function. The next step is to explore DNA-PK’s other RNA binding targets and the related molecular pathways.

“We’ve demonstrated that DNA-PK has an entirely separate role that has nothing to do with DNA repair,” Calo says. “In the future, we’re excited to learn what additional RNA-related duties it may have beyond stem cell maintenance.”

Top Image: Ribosomes are assembled in the nucleoli (shown here in human cells).

Citation:
“DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis”
Nature, online February 26, 2020, DOI: 10.1038/s41586-020-2041-2
Zhengping Shao, Ryan A. Flynn, Jennifer L. Crowe, Yimeng Zhu, Jialiang Liang, Wenxia Jiang, Fardin Aryan, Patrick Aoude, Carolyn R. Bertozzi, Verna M. Estes, Brian J. Lee, Govind Bhagat, Shan Zha, and Eliezer Calo