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KI Gallery Exhibit: Artifacts from a half century of cancer research

Celebrating 50 years of MIT's cancer research program and the individuals who have shaped its journey, the Koch Institute Gallery features 10 significant artifacts, from one of the earliest PCR machine developed by Nobel Laureate H. Robert Horvitz to a preserved zebrafish from the lab of Nancy Hopkins in the Koch Institute Public Galleries. Visit Monday through Friday, 9AM-5PM.

Koch Institute
November 21, 2024

Throughout 2024, MIT’s Koch Institute for Integrative Cancer Research has celebrated 50 years of MIT’s cancer research program and the individuals who have shaped its journey. In honor of this milestone anniversary year, on November 19, the Koch Institute celebrated the opening of a new exhibition: Object Lessons: Celebrating 50 Years of Cancer Research at MIT in 10 Items. Object Lessons invites the public to explore significant artifacts—from one of the earliest PCR machines, developed in the lab of Nobel laureate H. Robert Horvitz, to Greta, a groundbreaking zebrafish from the lab of Professor Nancy Hopkins—in the half century of discoveries and advancements that have positioned MIT at the forefront of the fight against cancer.

50 years of innovation

The exhibition provides a glimpse into the many contributors and advancements that have defined MIT’s cancer research history since the founding of the Center for Cancer Research in 1974. When the National Cancer Act was passed in 1971, very little was understood about the biology of cancer, and it aimed to deepen our understanding of cancer and develop better strategies for the prevention, detection, and treatment of the disease. MIT embraced this call to action, establishing a center where many leading biologists tackled cancer’s fundamental questions. Building on this foundation, the Koch Institute opened its doors in 2011, housing engineers and life scientists from many fields under one roof to accelerate progress against cancer in novel and transformative ways.

In the 13 years since, the Koch Institute’s collaborative and interdisciplinary approach to cancer research has yielded significant advances in our understanding of the underlying biology of cancer and allowed for the translation of these discoveries into meaningful patient impacts. Over 120 spin-out companies—many headquartered nearby in the Kendall Square area—have their roots in Koch Institute research, with nearly half having advanced their technologies to clinical trials or commercial applications. The Koch Institute’s collaborative approach extends beyond its labs: principal investigators often form partnerships with colleagues at world-renowned medical centers, bridging the gap between discovery and clinical impact.

Current Koch Institute Director Matthew Vander Heiden, also a practicing oncologist at the Dana-Farber Cancer Institute, is driven by patient stories.

“It is never lost on us that the work we do in the lab is important to change the reality of cancer for patients,” he says. “We are constantly motivated by the urgent need to translate our research and improve outcomes for those impacted by cancer.”

Symbols of progress

The items on display as part of Object Lessons take viewers on a journey through five decades of MIT cancer research, from the pioneering days of Salvador Luria, founding director of the Center for Cancer Research, to some of the Koch Institute’s newest investigators including Francisco Sánchez-Rivera, Eisen and Chang Career Development Professor and an assistant professor of biology, and Jessica Stark, Underwood-Prescott Career Development Professor and an assistant professor of biological engineering and chemical engineering.

Among the standout pieces is a humble yet iconic object: Salvador Luria’s ceramic mug, emblazoned with “Luria’s broth.” Lysogeny broth, often called—apocryphally—Luria Broth, is a medium for growing bacteria. Still in use today, the recipe was first published in 1951 by a research associate in Luria’s lab. The artifact, on loan from the MIT Museum, symbolizes the foundational years of the Center for Cancer Research and serves as a reminder of Luria’s influence as an early visionary. His work set the stage for a new era of biological inquiry that would shape cancer research at MIT for generations.

Visitors can explore firsthand how the Koch Institute continues to build on the legacy of its predecessors, translating decades of knowledge into new tools and therapies that have the potential to transform patient care and cancer research.

For instance, the PCR machine designed in the Horvitz Lab in the 1980s made genetic manipulation of cells easier, and gene sequencing faster and more cost-effective. At the time of its commercialization, this groundbreaking benchtop unit marked a major leap forward. In the decades since, technological advances have allowed for the visualization of DNA and biological processes at a much smaller scale, as demonstrated by the handheld BioBits® imaging device developed by Stark and on display next door to the Horvitz panel.

 “We created BioBits kits to address a need for increased equity in STEM education,” Stark says. “By making hands-on biology education approachable and affordable, BioBits kits are helping inspire and empower the next generation of scientists.”

While the exhibition showcases scientific discoveries and marvels of engineering, it also aims to underscore the human element of cancer research through personally significant items, such as a messenger bag and Seq-Well device belonging to Alex Shalek, J. W. Kieckhefer Professor in the Institute for Medical Engineering and Science and the Department of Chemistry.

Shalek investigates the molecular differences between individual cells, developing mobile RNA-sequencing devices. He could often be seen toting the bag around the Boston area, and worldwide as he perfected and shared his technology with collaborators near and far. Through his work, Shalek has helped to make single cell sequencing accessible for labs in more than 30 countries across six continents.

“The KI seamlessly brings together students, staff, clinicians, and faculty across multiple different disciplines to collaboratively derive transformative insights into cancer,” Shalek says. “To me, these sorts of partnerships are the best part about being at MIT.”

Around the corner from Shalek’s display, visitors will find an object that serves as a stark reminder of the real people impacted by Koch Institute research: Steven Keating’s SM’12, PhD ’16 3D-printed model of his own brain tumor. Keating, who passed away in 2019, became a fierce advocate for the rights of patients to their medical data, and came to know Vander Heiden through his pursuit to become an expert on his tumor type, IDH-mutant glioma. In the years since, Vander Heiden’s work has contributed to a new therapy to treat Steven’s tumor type. In 2024, the drug, called vorasidenib, gained FDA approval, providing the first therapeutic breakthrough for Keating’s cancer in more than 20 years.

As the Koch Institute looks to the future, Object Lessons stands as a celebration of the people, the science, and the culture that have defined MIT’s first half-century of breakthroughs and contributions to the field of cancer research.

“Working in the uniquely collaborative environment of the Koch Institute and MIT, I am confident that we will continue to unlock key insights in the fight against cancer,” says Vander Heiden. “Our community is poised to embark on our next 50 years with the same passion and innovation that has carried us this far.”

Object Lessons will be on view in the Koch Institute Public Galleries. Visit Monday through Friday, 9 a.m. to 5 p.m., to see the exhibit up close.

A blueprint for better cancer immunotherapies

By examining antigen architectures, MIT researchers built a therapeutic cancer vaccine that may improve tumor response to immune checkpoint blockade treatments.

Bendta Schroeder | Koch Institute
November 25, 2024

Immune checkpoint blockade (ICB) therapies can be very effective against some cancers by helping the immune system recognize cancer cells that are masquerading as healthy cells.

T cells are built to recognize specific pathogens or cancer cells, which they identify from the short fragments of proteins presented on their surface. These fragments are often referred to as antigens. Healthy cells will will not have the same short fragments or antigens on their surface, and thus will be spared from attack.

Even with cancer-associated antigens studding their surfaces, tumor cells can still escape attack by presenting a checkpoint protein, which is built to turn off the T cell. Immune checkpoint blockade therapies bind to these “off-switch” proteins and allow the T cell to attack.

Researchers have established that how cancer-associated antigens are distributed throughout a tumor determines how it will respond to checkpoint therapies. Tumors with the same antigen signal across most of its cells respond well, but heterogeneous tumors with subpopulations of cells that each have different antigens, do not. The overwhelming majority of tumors fall into the latter category and are characterized by heterogenous antigen expression. Because the mechanisms behind antigen distribution and tumor response are poorly understood, efforts to improve ICB therapy response in heterogenous tumors have been hindered.

In a new study, MIT researchers analyzed antigen expression patterns and associated T cell responses to better understand why patients with heterogenous tumors respond poorly to ICB therapies. In addition to identifying specific antigen architectures that determine how immune systems respond to tumors, the team developed an RNA-based vaccine that, when combined with ICB therapies, was effective at controlling tumors in mouse models of lung cancer.

Stefani Spranger, associate professor of biology and member of MIT’s Koch Institute for Integrative Cancer Research, is the senior author of the study, appearing recently in the Journal for Immunotherapy of Cancer. Other contributors include Koch Institute colleague Forest White, the Ned C. (1949) and Janet Bemis Rice Professor and professor of biological engineering at MIT, and Darrell Irvine, professor of immunology and microbiology at Scripps Research Institute and a former member of the Koch Institute.

While RNA vaccines are being evaluated in clinical trials, current practice of antigen selection is based on the predicted stability of antigens on the surface of tumor cells.

“It’s not so black-and-white,” says Spranger. “Even antigens that don’t make the numerical cut-off could be really valuable targets. Instead of just focusing on the numbers, we need to look inside the complex interplays between antigen hierarchies to uncover new and important therapeutic strategies.”

Spranger and her team created mouse models of lung cancer with a number of different and well-defined expression patterns of cancer-associated antigens in order to analyze how each antigen impacts T cell response. They created both “clonal” tumors, with the same antigen expression pattern across cells, and “subclonal” tumors that represent a heterogenous mix of tumor cell subpopulations expressing different antigens. In each type of tumor, they tested different combinations of antigens with strong or weak binding affinity to MHC.

The researchers found that the keys to immune response were how widespread an antigen is expressed across a tumor, what other antigens are expressed at the same time, and the relative binding strength and other characteristics of antigens expressed by multiple cell populations in the tumor

As expected, mouse models with clonal tumors were able to mount an immune response sufficient to control tumor growth when treated with ICB therapy, no matter which combinations of weak or strong antigens were present. However, the team discovered that the relative strength of antigens present resulted in dynamics of competition and synergy between T cell populations, mediated by immune recognition specialists called cross-presenting dendritic cells in tumor-draining lymph nodes. In pairings of two weak or two strong antigens, one resulting T cell population would be reduced through competition. In pairings of weak and strong antigens, overall T cell response was enhanced.

In subclonal tumors, with different cell populations emitting different antigen signals, competition rather than synergy was the rule, regardless of antigen combination. Tumors with a subclonal cell population expressing a strong antigen would be well-controlled under ICB treatment at first, but eventually parts of the tumor lacking the strong antigen began to grow and developed the ability evade immune attack and resist ICB therapy.

Incorporating these insights, the researchers then designed an RNA-based vaccine to be delivered in combination with ICB treatment with the goal of strengthening immune responses suppressed by antigen-driven dynamics. Strikingly, they found that no matter the binding affinity or other characteristics of the antigen targeted, the vaccine-ICB therapy combination was able to control tumors in mouse models. The widespread availability of an antigen across tumor cells determined the vaccine’s success, even if that antigen was associated with weak immune response.

Analysis of clinical data across tumor types showed that the vaccine-ICB therapy combination may be an effective strategy for treating patients with tumors with high heterogeneity. Patterns of antigen architectures in patient tumors correlated with T cell synergy or competition in mice models and determined responsiveness to ICB in cancer patients. In future work with the Irvine laboratory at the Scripps Research Institute, the Spranger laboratory will further optimize the vaccine with the aim of testing the therapy strategy in the clinic.

Whitehead Institute Member Sebastian Lourido receives the 2024 William Trager Award

Sebastian Lourido was awarded the 2024 William Trager Award by the American Society of Tropical Medicine and Hygiene for his pioneering use of CRISPR tools to study the biology of Toxoplasma gondii, a single-celled parasite that infects about 25% of humans.

Merrill Meadow | Whitehead Institute
November 14, 2024

The Trager Award recognizes scientists who have made substantial contributions to the study of basic parasitology through breakthroughs that have unlocked completely new areas of work.

ASTMH selected Lourido — who is also an associate professor of Biology at Massachusetts Institute of Technology and holds the Landon Clay Career Development Chair at Whitehead Institute — in recognition of his groundbreaking discoveries on the molecular biology of Toxoplasma. In particular, Lourido has been lauded for his use of cutting-edge CRISPR tools to study the fundamental biology of Toxoplasma gondii, a single-celled parasite that infects about 25 percent of humans.

“My laboratory colleagues and I are grateful for this recognition of our work, and for the wonderful opportunity it presents to more widely share the ideas and tools we have developed,” says Lourido, who will deliver a talk on his research at the ASTMH Annual Meeting in New Orleans on Nov. 15, 2024.

Research findings: Open technology platform enables new versatility for neuroscience research with more naturalistic behavior

System developed by MIT, including co-author Mathew Wilson, and Open Ephys team provides a fast, light, standardized means for combining multiple instruments with minimal hindrance of lab mouse mobility.

David Orenstein | The Picower Institute for Learning and Memory
November 13, 2024

Individual technologies for recording and controlling neural activity in the brains of research mice have each advanced rapidly but the potential of easily mixing and matching them to conduct more sophisticated experiments, all while enabling the most natural behavior possible, has been difficult to realize. To empower a new generation of neuroscience experiments, engineers and scientists at MIT and the Open Ephys cooperative have developed a new standardized, open-source hardware and software platform. They described the system, called ONIX, in a new study Nov. 11 in Nature Methods.

ONIX provides labs with a means to acquire data simultaneously from multiple popular implanted technologies (such as electrodes, microscopes and stimulation probes) while also powering and controlling those independent devices via a very thin coaxial cable and unimposing headstage. The system provides a standardized means of acquiring each instrument’s data and neatly integrating it all for efficient transmission to desktop software where scientists can then see and work with it. In the study the researchers document ONIX’s high data throughput and low latency. They also demonstrate that because the system’s headstage and cable are so physically light and resistant to twisting, mice can behave completely naturally and wear the system for days on end. In a large enclosure at MIT with a complex 3D landscape, for instance, mice wearing the system were able to nimbly scamper, climb and leap in experiments comparably to mice wearing no hardware at all.

“ONIX represents the culmination of many quantitative improvements that all come together to enable a qualitative leap in our ability to perform neural recordings in naturalistic behavior,” said corresponding author Jakob Voigts, an MIT neuroscience alumnus, co-founder of Open Ephys, and a research group leader at the Janelia Research Campus of the Howard Hughes Medical Institute. “We can now study the brain during behaviors that unfold over many hours and allow the animals to learn, to make a lot of complex decisions, and to interact with the world in ways that were previously not accessible.”

Jon Newman, a former MIT postdoc and now president of Open Ephys, and MIT postdoc Jie “Jack” Zhang led the work in the lab of co-author Matt Wilson, Sherman Fairchild Professor in The Picower Institute for Learning and Memory at MIT, together with Aarón Cuevas-López at Open Ephys. Wilson, whose lab studies neural processes underlying memory, said the idea behind developing ONIX was to develop a set of standards that would make it easy for any lab to use multiple technologies to acquire rich neural data while animals performed complex behaviors over long time periods.

“Jon’s motivation, the principle he used, was that if we need to do experiments that combined things like optogenetics, imaging, tetrode electrophysiology, and neuropixels, could we do it in a way that would not only enable experiments we were doing but also more complex experiments, involving more complex behavior, involving the integration of different recording methodologies that advances the whole community and not just one individual lab?,” said Wilson, a faculty member in MIT’s Departments of Biology and Brain and Cognitive Sciences (BCS).

Open origins

As Newman and then Zhang began to develop the technology starting in 2016 with this community-minded, open-source philosophy, Wilson said, it was natural to do so in partnership with Open Ephys, an MIT-born effort, now based in Atlanta, which develops and disseminates open, standardized systems to for neuroscience research. Making systems open-source provides researchers with many advantages, Voigts explained.

“Anyone can download the plans for the hardware as well as the software that make up the system,” Voigts said. “For technically well-versed neuroscientists this means that it is easier to modify aspects of the system. Open source also means that the system works with probes from many manufacturers because the connectors and standards aren’t proprietary. Most importantly, the open standards and design allow hardware and software developers to use ONIX as a starting point for completely new tools.”

Voigts compared ONIX to the USB standard people enjoy on their computers and phones. Any number of accessories can easily work with those devices because all they have to do is plug in. Similarly with ONIX, Wilson said, “You can mix and match and combine and then add new technologies without having to re-engineer the whole system.”

Lab demos

To validate the platform, the researchers conducted several experiments with mice including in Wilson’s lab and in the lab of co-author Mark Harnett, Associate Professor in the McGovern Institute for Brain Research and BCS Department at MIT (where Voigts did his postdoctoral work).

In their experiments they compared the mobility of mice implanted with electrodes but sometimes wearing ONIX (and its 0.3 mm tether cable) vs. sometimes wearing a commonly used and but substantially thicker (1.8 mm) tether cable over an 8-hour neural recording session. The mice proved to be much more mobile while wearing the lighter and thinner ONIX system, showing a broader range of exploration, freer head movement, and much faster running speeds. In a similar experiment in which mice were inplanted with tetrodes in the brain’s retrosplenial cortex, they even were able to jump while wearing ONIX but did not while wearing the more imposing tether. In another experiment the researchers compared mouse mobility around the enclosure between ONIX-wearing and completely unimplanted mice. The mice explored with equal freedom (as measured by motion tracking cameras) though the ONIX mice didn’t run as fast as unimplanted mice.

In further experiments, Voigts’s team at Janelia used ONIX to record for 55 hours because the system kept its cable tangle-free over that long-duration activity.

Finally the researchers showed that ONIX could transmit recordings not only from implanted electrodes and tetrodes but also from miniscopes and neuropixels, via experiments at the Allen Institute for Brain Science. They also showed how Open Ephys’s data acquisition software Bonsai (developed by co-author Goncalo Lopes) enabled the brain activity recordings to be synchronized with behavior tracking cameras to correlate neural activity and behavior.

Voigts said he hopes the system earns widespread adoption, especially as hardware costs continue to come down.

“I hope that this system convinces others to take the plunge and record neural data in more complex animal behaviors,” he said.

In addition to the authors named above, other authors are Nicholas Miller, Takato Honda, Marie-Sophie van der Goes, Alexandra Leighton Felipe Carvalho, Anna Lakunina, and Joshua Siegle, who co-founded Open Ephys with Voigts.

Funding for the study came from the National Institutes of Health, The Picower Institute for Learning and Memory, The JPB Foundation, the National Science Foundation, a Brain Science Foundation Research Grant Award, a Kavli-Grass-MBL Fellowship by the Kavli Foundation, the Grass Foundation, and Marine Biological Laboratory (MBL), an Osamu Hayaishi Memorial Scholarship for Study Abroad, a Uehara Memorial Foundation Overseas Fellowship, and Japan Society for the Promotion of Science (JSPS) Overseas Fellowship. a Mathworks Graduate Fellowship. The Simons Center for the Social Brain at MIT and the Howard Hughes Medical Institute.

Alumni Spotlight: Distillery Founder with a Spirited Passion

Jennifer Yang, '97, has been drawing on her biology degree for making spirits at a craft distillery in Maryland.

Jessica R. Simpson | Slice of MIT
October 15, 2024

If you had told Jennifer Yang ’97 during her time as a Course 7 major at MIT that she would use her biology degree to run a distillery, she wouldn’t have believed you.

“When I was at MIT, I looked at entrepreneurs and I thought, ‘Oh my gosh, that’s not me. I’m not one of those people who are so innovative and gutsy and brave,’” Yang says.

Managing a distillery is a passion that matured in Yang over time—much like the complex flavor of a barrel-aged whiskey. After graduating from MIT, the New York-native moved to Washington, DC, to pursue a career in management and technology consulting, which involved a lot of after-hours networking events. While building connections with colleagues over a glass of whiskey—a drink that was particularly popular with clients—Yang discovered her passion. Over the course of 10 years, she researched the science of making spirits, explored different small distilleries, and even started a whiskey tasting club.

“Being a science geek at heart and being very curious, I went down this rabbit hole pretty quickly in terms of wanting to learn more about it,” Yang explains.

In November 2022, she and her husband opened Covalent Spirits, a craft distillery, tasting room, and event space in Westminster, Maryland. In addition to producing bourbon whiskey, Covalent Spirits distills and blends vodka, gin, rum, and liqueurs. One of the bar’s unique and in-demand offerings is the “pH,” or “power of hydrogen,” cocktail, which uses the acidity of lemonade to turn a blue tea into a vibrant purple. Yang still works in consulting, but you can find her in her element behind the bar, engineering “pH” (and many other) cocktails Thursday through Saturday.

In her spare time, Yang is a committed MIT volunteer. An active participant in the Club of Washington DC, she is the regional alumni ambassador for the Baltimore area as well. Yang is also an educational counselor and the current president of the Class of 1997. She notes that she and her ’97 classmates were the first to organize pi reunions, a tradition in which alums gather in Las Vegas 3.14 years after graduation. “We’re glad our class could leave a little bit of a legacy,” she says.

In fact, the shared MIT connection between alumni inspired Yang to name her company Covalent Spirits. One year, at an MIT gathering, Yang started talking to another alum about planning events for undergrad classes that shared years at MIT—what they called “covalent classes.” Yang has since incorporated literal and metaphorical covalent bonds (a chemical connection between atoms formed by sharing) into every facet of her business: from the chemistry of making spirits, to the design of the distillery logo, to the company’s emphasis on community.

“While we are striving to create really good products, we also want to create a space and experiences for people to get together and geek out over a common interest, to celebrate an occasion, or to connect over anything,” Yang elaborates. “You share a drink, you share an experience, you share a community. Bonding through sharing is the covalent spirit.”

PNAS Profile: Catherine Drennan

Structural intuitions lead to structural insights

Jennifer Viegas | PNAS
November 8, 2024

HHMI Investigator and Professor of Biology and Chemistry Catherine Drennan has spent a distinguished career addressing challenging and wide-ranging structural biology problems.

Catherine Drennan, a Howard Hughes Medical Institute investigator and professor and a professor of biology and chemistry at the Massachusetts Institute of Technology (MIT), has spent a distinguished career addressing challenging and wide-ranging structural biology problems. These include her discovery, while she was a graduate student, of the structure of vitamin B12 bound to protein and her recent determination at atomic resolution of the structure of an active ribonucleotide reductase (RNR) with water molecules, findings reported in her Inaugural Article (IA) (1).

Drennan, who was elected to the National Academy of Sciences in 2023, has uncovered the form and function of metalloenzymes that use metal cofactors to catalyze chemical reactions involving free radicals. Metalloenzymes are of broad human health and environmental interest; some are promising antibiotic and cancer drug targets, whereas others hold the potential for bioremediation efforts, such as converting carbon dioxide into biofuels.

Family of Accomplished Scientists

Drennan was raised in New York City by her father, an obstetrician–gynecologist, and her mother, an anthropologist. Her father was born in Germany and attended medical school at the University of Hamburg. Harboring antifascist leanings, he fled Germany in 1933. He completed medical training in Geneva, Switzerland, before obtaining political asylum in 1940 in the United States, where he became one of the first doctors to practice the Lamaze Method of natural childbirth.
Drennan’s mother attended Antioch College, where she was a student of civil engineer Arthur Ernest Morgan, who was appointed in 1948 to India’s first University Education Commission. She accompanied Morgan to India and served as his administrative assistant before earning her doctorate in anthropology at Cornell University.

“Both my parents were endlessly curious,” says Drennan. “My father was fascinated by the molecular basis of medicine, and my mother was fascinated by people and instilled in me her love for storytelling, teaching, and mentoring.”

Diagnosed with Dyslexia

Although she was an attentive student, Drennan did not learn to read until her second time through sixth grade. “When I finally learned how to read, it was by memorizing the shapes of words,” says Drennan, who was diagnosed with dyslexia when she was in first grade. “Over time I became very good at shape recognition. I am not disabled; I am differently abled. The skill set that I developed to compensate for my dyslexia has made me a world-class structural biologist. We all have strengths and weaknesses, and my ‘weakness’ is also my superpower.”
She was accepted to Vassar College, where she earned a bachelor’s degree in chemistry in 1985. “Miriam Rossi was my undergraduate chemistry research advisor, and she believed in me before I believed in me,” Drennan says. Upon Rossi’s advice, Drennan pursued a doctorate, but not before teaching high-school science and drama at Scattergood Friends School in Iowa.
Following three years of high-school teaching, Drennan pursued graduate studies at the University of Michigan, Ann Arbor, where she earned a PhD in 1995, served as a research fellow from 1995 to 1996, and was mentored by biochemists Martha Ludwig and Rowena Matthews. “They treated me as a colleague, allowing me to see myself as a scientist of value,” Drennan says. “I learned so much from these two incredible scientists. They are, and always will be, my heroes.”

Structure of Vitamin B12 Bound to Protein

With Ludwig et al., Drennan determined the structure of cobalamin (vitamin B12) bound to protein (2). This crystal structure revealed how the protein modulates the reactivity of the B12 cofactor to enable its critical roles in metabolism.
From 1996 to 1999, Drennan did a postdoctoral stint at the California Institute of Technology, under the mentorship of structural biologist Douglas Rees. “Doug taught by example that one does not have to be cutthroat to succeed in the competitive area of structural biology,” she says. “He has continued to mentor me throughout my career, helping me through challenging times.”
Another important mentor was chemist JoAnne Stubbe, a leader in the study of RNRs who recruited Drennan to MIT in 1999 as an assistant professor of chemistry and has been her collaborator for the past 25 years. Drennan says, “Her passion for scientific discovery is unmatched and has inspired me to keep digging to try to understand, at the most fundamental level, how ribonucleotide reductase works.” Drennan advanced to an associate professorship at MIT in 2004 and a full professorship in 2006.

Revealing Metalloenzyme Form and Function

Drennan’s group continues to study B12 and has provided numerous snapshots of cobalamin-dependent proteins and protein complexes. The findings have changed what is known about B12 functions and mechanisms. Using X-ray crystallography, the researchers unveiled a protein complex capable of methyl transfer from folate to B12 (3). They obtained snapshots of the biological process involved in loading B12 into an enzyme (4) and provided structural data on how B12 can be repurposed from enzyme cofactor to light sensor (5).
Drennan has also worked on uncovering the structures of enzymes containing radical S-adenosylmethionine (SAM) cofactors. Drennan and colleagues revealed an X-ray structure of a radical SAM enzyme (6), helping to establish the “core” fold for an enzyme superfamily that has over 100,000 members. Her group further elucidated structures of SAM family members with functions including posttranslational modification (7), antibiotic and antiviral compound biosynthesis (8, 9), and vitamin biosynthesis (6, 10).
Mononuclear nonheme iron enzymes are also of interest to Drennan. The cofactor is simple, but the reactions catalyzed are complex. Her group reported the structure of a nonheme iron halogenase, showing that the halogen binds directly to the catalytic iron (11). Drennan says, “This was a complete surprise that required new mechanistic proposals to be written.”

“Oceanic Methane Paradox”

Early in her independent career, Drennan determined one of the first structures of a nickel-iron-sulfur-dependent carbon monoxide dehydrogenase (CODH), which plays an important role in the global carbon cycle (12). The structure, along with that of an associated enzyme complex (13), provided a series of snapshots of the multiple metal ion centers underlying the ability of certain microbes to live off hydrogen gas and carbon dioxide in a process known as acetogenesis. More recently, they investigated the molecular basis by which the activity of CODH enzymes can be restored after oxygen exposure (14), a discovery with implications for the industrial use of CODHs.
Drennan and her team have also studied the organic compound methylphosphonate that was proposed as a source of methane from the aerobic upper ocean; the biological source was long a mystery. When a methylphosphonate synthase was discovered by chemical biologist Wilfred van der Donk and coworkers, part of the mystery was solved but the gene for this enzyme did not appear to be widespread. When Drennan and colleagues solved the structure of a methylphosphonate synthase; however, they discovered a sequence motif showing that the gene was, in fact, abundant in microbes that inhabit the upper ocean (15). This seminal finding is credited with resolving the oceanic methane paradox.

Radical-Based Chemistry in Ribonucleotide Reductases

Human RNR is an established chemotherapeutic target, and bacterial RNRs hold promise as antibiotic targets. So Drennan and her team have a longstanding interest in uncovering the mechanisms of RNRs. In 2002, her lab determined the structure of a B12-dependent RNR, which showed how cobalamin could be used to initiate radical chemistry (16). Nearly a decade later, Drennan’s team revealed how high levels of the nucleotide deoxyadenosine triphosphate (dATP) down-regulate RNR activity (17, 18). They subsequently provided structures showing the molecular basis of allosteric specificity, which maintains the proper ratios of RNA to DNA building blocks (19), and demonstrated the importance of RNR activity regulation (20).
An atomic-resolution structure of any RNR in an active state had been elusive for many years. Drennan and her team achieved the feat in 2020 when they trapped the active state of Escherichia coli RNR and determined its structure by cryoelectron microscopy (21). However, the resolution of the structure was too low for the visualization of water molecules believed to be critical in the radical transfer pathway.
In her IA, Drennan (1) describes how her team resolved the problem, presenting the structure of an active RNR at atomic resolution allowing for the visualization of water molecules. She explains, “This time, instead of using unnatural amino acids to trap the structure, we used a mechanism-based inhibitor. It was a very long road to get to these data, but it was worth the wait.”

“Superheroes of the Cell”

For her achievements, Drennan has received MIT’s Everett Moore Baker Memorial Award for Excellence in Undergraduate Teaching (2005, 2024), the Dorothy Crowfoot Hodgkin Award from The Protein Society (2020), and the William C. Rose Award from the American Society for Biochemistry and Molecular Biology (2023), among other honors. She has mentored nearly 100 undergraduates and dozens of graduate students and postdoctoral associates, many of whom are from underrepresented minority groups or disadvantaged backgrounds. She considers her students extended family members and takes pride in their accomplishments.
She and her team continue to work on RNR using the tools of structural biology. She says, “We want to obtain a deeper level of understanding of the human RNR, which is a cancer drug target. We also want to identify differences between the human enzyme and bacterial RNRs, differences that could be exploited in the development of new antibiotics.”
Beyond these efforts, Drennan’s overall goal is to understand how enzymes control radical species to enable challenging chemical reactions without damaging themselves or their cellular environment. “Radical enzymes are like the Avengers, powerful but with a high potential for collateral damage,” she explains. “I am fascinated by how nature catalyzes the most challenging of chemical reactions. The enzymes that do this work are the superheroes of the cell and I want to know their secrets.”
1.
D. E. Westmoreland et al., 2.6-Å resolution cryo-EM structure of a class Ia ribonucleotide reductase trapped with mechanism-based inhibitor N3CDP. Proc. Natl. Acad. Sci. U.S.A. 121, e2417157121 (2024). Crossref. PubMed.
2.
C. L. Drennan et al., How a protein binds B12: A 3.0 Å X-ray structure of B12-binding domains of methionine synthase. Science 266, 1669–1674 (1994). Crossref. PubMed.
3.
Y. Kung et al., Visualizing molecular juggling within a B12-dependent methyltransferase complex. Nature 484, 265–269 (2012). Crossref. PubMed.
4.
F. A. Vaccaro, D. A. Born, C. L. Drennan, Structure of metallochaperone in complex with the cobalamin-binding domain of its target mutase provides insight into cofactor delivery. Proc. Natl. Acad. Sci. U.S.A. 120, e2214085120 (2023). Crossref. PubMed.
5.
M. Jost et al., Structural basis for gene regulation by a B12-dependent photoreceptor. Nature 526, 536–541 (2015). Crossref. PubMed.
6.
F. Berkovitch et al., Crystal structure of biotin synthase, an S-Adenosylmethionine-dependent radical enzyme. Science 303, 76–79 (2004). Crossref. PubMed.
7.
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A new approach to modeling complex biological systems

MIT engineers’ new model could help researchers glean insights from genomic data and other huge datasets. This is potentially critical to researchers who study any kind of complex biological system, according to senior author Douglas Lauffenburger.

Anne Trafton | MIT News
November 5, 2024

Over the past two decades, new technologies have helped scientists generate a vast amount of biological data. Large-scale experiments in genomics, transcriptomics, proteomics, and cytometry can produce enormous quantities of data from a given cellular or multicellular system.

However, making sense of this information is not always easy. This is especially true when trying to analyze complex systems such as the cascade of interactions that occur when the immune system encounters a foreign pathogen.

MIT biological engineers have now developed a new computational method for extracting useful information from these datasets. Using their new technique, they showed that they could unravel a series of interactions that determine how the immune system responds to tuberculosis vaccination and subsequent infection.

This strategy could be useful to vaccine developers and to researchers who study any kind of complex biological system, says Douglas Lauffenburger, the Ford Professor of Engineering in the departments of Biological Engineering, Biology, and Chemical Engineering.

“We’ve landed on a computational modeling framework that allows prediction of effects of perturbations in a highly complex system, including multiple scales and many different types of components,” says Lauffenburger, the senior author of the new study.

Shu Wang, a former MIT postdoc who is now an assistant professor at the University of Toronto, and Amy Myers, a research manager in the lab of University of Pittsburgh School of Medicine Professor JoAnne Flynn, are the lead authors of a new paper on the work, which appears today in the journal Cell Systems.

Modeling complex systems

When studying complex biological systems such as the immune system, scientists can extract many different types of data. Sequencing cell genomes tells them which gene variants a cell carries, while analyzing messenger RNA transcripts tells them which genes are being expressed in a given cell. Using proteomics, researchers can measure the proteins found in a cell or biological system, and cytometry allows them to quantify a myriad of cell types present.

Using computational approaches such as machine learning, scientists can use this data to train models to predict a specific output based on a given set of inputs — for example, whether a vaccine will generate a robust immune response. However, that type of modeling doesn’t reveal anything about the steps that happen in between the input and the output.

“That AI approach can be really useful for clinical medical purposes, but it’s not very useful for understanding biology, because usually you’re interested in everything that’s happening between the inputs and outputs,” Lauffenburger says. “What are the mechanisms that actually generate outputs from inputs?”

To create models that can identify the inner workings of complex biological systems, the researchers turned to a type of model known as a probabilistic graphical network. These models represent each measured variable as a node, generating maps of how each node is connected to the others.

Probabilistic graphical networks are often used for applications such as speech recognition and computer vision, but they have not been widely used in biology.

Lauffenburger’s lab has previously used this type of model to analyze intracellular signaling pathways, which required analyzing just one kind of data. To adapt this approach to analyze many datasets at once, the researchers applied a mathematical technique that can filter out any correlations between variables that are not directly affecting each other. This technique, known as graphical lasso, is an adaptation of the method often used in machine learning models to strip away results that are likely due to noise.

“With correlation-based network models generally, one of the problems that can arise is that everything seems to be influenced by everything else, so you have to figure out how to strip down to the most essential interactions,” Lauffenburger says. “Using probabilistic graphical network frameworks, one can really boil down to the things that are most likely to be direct and throw out the things that are most likely to be indirect.”

Mechanism of vaccination

To test their modeling approach, the researchers used data from studies of a tuberculosis vaccine. This vaccine, known as BCG, is an attenuated form of Mycobacterium bovis. It is used in many countries where TB is common but isn’t always effective, and its protection can weaken over time.

In hopes of developing more effective TB protection, researchers have been testing whether delivering the BCG vaccine intravenously or by inhalation might provoke a better immune response than injecting it. Those studies, performed in animals, found that the vaccine did work much better when given intravenously. In the MIT study, Lauffenburger and his colleagues attempted to discover the mechanism behind this success.

The data that the researchers examined in this study included measurements of about 200 variables, including levels of cytokines, antibodies, and different types of immune cells, from about 30 animals.

The measurements were taken before vaccination, after vaccination, and after TB infection. By analyzing the data using their new modeling approach, the MIT team was able to determine the steps needed to generate a strong immune response. They showed that the vaccine stimulates a subset of T cells, which produce a cytokine that activates a set of B cells that generate antibodies targeting the bacterium.

“Almost like a roadmap or a subway map, you could find what were really the most important paths. Even though a lot of other things in the immune system were changing one way or another, they were really off the critical path and didn’t matter so much,” Lauffenburger says.

The researchers then used the model to make predictions for how a specific disruption, such as suppressing a subset of immune cells, would affect the system. The model predicted that if B cells were nearly eliminated, there would be little impact on the vaccine response, and experiments showed that prediction was correct.

This modeling approach could be used by vaccine developers to predict the effect their vaccines may have, and to make tweaks that would improve them before testing them in humans. Lauffenburger’s lab is now using the model to study the mechanism of a malaria vaccine that has been given to children in Kenya, Ghana, and Malawi over the past few years.

“The advantage of this computational approach is that it filters out many biological targets that only indirectly influence the outcome and identifies those that directly regulate the response. Then it’s possible to predict how therapeutically altering those biological targets would change the response. This is significant because it provides the basis for future vaccine and trial designs that are more data driven,” says Kathryn Miller-Jensen, a professor of biomedical engineering at Yale University, who was not involved in the study.

Lauffenburger’s lab is also using this type of modeling to study the tumor microenvironment, which contains many types of immune cells and cancerous cells, in hopes of predicting how tumors might respond to different kinds of treatment.

The research was funded by the National Institute of Allergy and Infectious Diseases.

Sauer & Davis Lab News Brief: structures of molecular woodchippers reveal mechanism for versatility

Rest in pieces: deconstructing polypeptide degradation machinery

Lillian Eden | Department of Biology
November 12, 2024

Research from the Sauer and Davis Labs in the Department of Biology at MIT shows that conformational changes contribute to the specificity of “molecular woodchippers” 

Degradation is a crucial process for maintaining protein homeostasis by culling excess or damaged proteins whose components can then be recycled. It is also a highly regulated process—for good reason. A cell could potentially waste many resources if the degradation machinery destroys proteins it shouldn’t. 

One of the major pathways for protein degradation in bacteria and eukaryotic mitochondria involves a molecular machine called ClpXP. ClpXP is made up of two components: a star-shaped structure made up of six subunits called ClpX that engages and unfolds proteins tagged for degradation, and an associated barrel-shaped enzyme, called ClpP, that chemically breaks up proteins into small pieces called peptides. 

ClpXP is incredibly adaptable and is often compared to a woodchipper — able to take in materials and spit out their broken-down components. Thanks to biochemical experiments, this molecular degradation machine is known to be able to break down hundreds of different proteins in the cell regardless of physical or chemical properties such as size, shape, or charge. ClpX uses energy from ATP hydrolysis to unfold proteins before they are threaded through its central channel, referred to as the axial channel, and into the degradation chamber of ClpP.

In three papers, one in PNAS and two in Nature Communications, researchers from the Department of Biology at MIT have expanded our understanding of how this molecular machinery engages with, unfolds, and degrades proteins — and how that machinery refrains, by design, from unfolding proteins not tagged for degradation. 

Alireza Ghanbarpour, until recently a postdoc in the Sauer Lab and Davis Lab and first author on all three papers, began with a simple question: given the vast repertoire of potential substrates — that is, proteins to be degraded — how is ClpXP so specific?

Ghanbarpour — now an assistant professor in the Department of Biochemistry and Molecular Biology at Washington University School of Medicine in St. Louis — found that the answer to this question lies in conformational changes in the molecular machine as it engages with an ill-fated protein. 

Reverse Engineering using Structural Insights

Ghanbarpour approached the question of ClpXP’s versatility by characterizing conformational changes of the molecular machine using a technique called cryogenic electron microscopy. In cryo-EM, sample particles are frozen in solution, and images are collected; algorithms then create 3D renderings from the 2D images.

“It’s really useful to generate different structures in different conditions and then put them together until you know how a machine works,” he says. “I love structural biology, and these molecular machines make fascinating targets for structural work and biochemistry. Their structural plasticity and precise functions offer exciting opportunities to understand how nature leverages enzyme conformations to generate novel functions and tightly regulate protein degradation within the cell.”

Inside the cell, these proteases do not work alone but instead work together with “adaptor” proteins, which can promote — or inhibit — degradation by ClpXP. One of the adaptor proteins that promotes degradation by ClpXP is SspB. 

In E. coli and most other bacteria, ClpXP and SspB interact with a tag called ssrA that is added to incomplete proteins when their biosynthesis on ribosomes stalls. 

The tagging process frees up the ribosome to make more proteins, but creates a problem: incomplete proteins are prone to aggregation, which could be detrimental to cellular health and can lead to disease. By interacting with the degradation tag, ClpXP and SspB help to ensure the degradation of these incomplete proteins. Understanding this process and how it may go awry may open therapeutic avenues in the future.

“It wasn’t clear how certain adapters were interacting with the substrate and the molecular machines during substrate delivery,” Ghanbarpour notes. “My recent structure reveals that the adapter engages with the enzyme, reaching deep into the axial channel to deliver the substrate.” 

Ghanbarpour and colleagues showed that ClpX engages with both the SspB adaptor and the ssrA degradation tag of an ill-fated protein at the same time. Surprisingly, they also found that this interaction occurs while the upper part of the axial channel through ClpX is closed — in fact, the closed channel allows ClpX to contact both the tag and the adaptor simultaneously.

This result was surprising, according to senior author and Salvador E. Luria Professor of Biology Robert Sauer, whose lab has been working on understanding this molecular machine for more than two decades: it was unclear whether the channel through ClpX closes in response to a substrate interaction, or if the channel is always closed until it opens to pass an unfolded protein down to ClpP to be degraded.

Preventing Rogue Degradation

Throughout this project, Ghanbarpour was co-advised by structural biologist and Associate Professor of Biology Joey Davis and collaborated with members of the Davis Lab to better understand the conformational changes that allow these molecular machines to function. Using a cryo-EM analysis approach developed in the Davis lab called CryoDRGN, the researchers showed that there is an equilibrium between ClpXP in the open and closed states: it’s usually closed but is open in about 10% of the particles in their samples. 

The closed state is almost identical to the conformation ClpXP assumes when it is engaged with an ssrA-tagged substrate and the SspB adaptor. 

To better understand the biological significance of this equilibrium, Ghanbarpour created a mutant of ClpXP that is always in the open position. Compared to normal ClpXP, the mutant degraded some proteins lacking obvious degradation tags faster but degraded ssrA-tagged proteins more slowly. 

According to Ghanbarpour, these results indicate that the closed channel improves ClpXP’s ability to efficiently engage tagged proteins meant to be degraded, whereas the open channel allows more “promiscuous” degradation. 

Pausing the Process

The next question Ghanbarpour wanted to answer was what this molecular machine looks like while engaged with a protein it is attempting to unfold. To do that, he created a substrate with a highly stable protein attached to the degradation tag that is initially pulled into ClpX, but then dramatically slows protein unfolding and degradation.

In the structures where the degradation process stalls, Ghanbarpour found that the degradation tag was pulled far into the molecular machine—through ClpX and into ClpP—and the folded protein part of the substrate was pulled tightly against the axial channel of ClpX. 

The opening of the axial channel, called the axial pore, is made up of looping protein structures called RKH loops. These flexible loops were found to play roles both in recognizing the ssrA degradation tag and in how substrates or the SspB adaptor interact with or are pulled against the channel during degradation. 

The flexibility of these RKH loops allows ClpX to interact with a large number of different proteins and adapters, and these results clarify some previous biochemical and mutational studies of interactions between the substrate and ClpXP. 

Although Ghanbarpour’s recent work focused on just one adaptor and degradation tag, he noted there are many more targets — ClpXP is something akin to a Swiss army knife for breaking down polypeptide chains. 

The way those other substrates interact with ClpXP could differ from the structures solved with the SspB adaptor and ssrA tag. It also stands to reason that the way ClpXP reacts to each substrate may be unique. For example, given that ClpX is occasionally in an open state, some substrates may engage with ClpXP only while it’s in an open conformation. 

In his new position at Washington University, Ghanbarpour intends to continue exploring how ClpXP and other molecular machines locate their target substrates and interact with adaptors, shedding light on how cells regulate protein degradation and maintain protein homeostasis.

The structures Ghanbarpour solved involved free-floating protein degradation machinery, but membrane-bound degradation machinery also exists. The membrane-bound version’s structure and conformational adaptions potentially differ from the structures Ghanbarpour found in his previous three papers. Indeed, in a recent preprint, Ghanbarpour worked on the cryo-EM structure of a nautilus shell-shaped protein assembly that seems to control membrane-bound degradation machinery. This assembly plays a critical role in regulating protein degradation within the bacterial inner membrane.

“The function of these proteases goes beyond simply degrading damaged proteins. They also target transcription factors, regulatory proteins, and proteins that don’t exist in normal conditions,” he says. “My new lab is particularly interested in understanding how cells use these proteases and their accessory adaptors, both under normal and stress conditions, to reshape the proteome and support recovery from cellular distress.”

A cell protector collaborates with a killer

New research from the Horvitz Lab reveals what it takes for a protein that is best known for protecting cells against death to take on the opposite role.

Jennifer Michalowski | McGovern Institute
November 1, 2024

From early development to old age, cell death is a part of life. Without enough of a critical type of cell death known as apoptosis, animals wind up with too many cells, which can set the stage for cancer or autoimmune disease. But careful control is essential, because when apoptosis eliminates the wrong cells, the effects can be just as dire, helping to drive many kinds of neurodegenerative disease.

By studying the microscopic roundworm Caenorhabditis elegans—which was honored with its fourth Nobel Prize last month—scientists at MIT’s McGovern Institute have begun to unravel a longstanding mystery about the factors that control apoptosis: how a protein capable of preventing programmed cell death can also promote it. Their study, led by McGovern Investigator Robert Horvitz and reported October 9, 2024, in the journal Science Advances, sheds light on the process of cell death in both health and disease.

“These findings, by graduate student Nolan Tucker and former graduate student, now MIT faculty colleague, Peter Reddien, have revealed that a protein interaction long thought to block apoptosis in C. elegans, likely instead has the opposite effect,” says Horvitz, who shared the 2002 Nobel Prize for discovering and characterizing the genes controlling cell death in C. elegans.

Mechanisms of cell death

Horvitz, Tucker, Reddien and colleagues have provided foundational insights in the field of apoptosis by using C. elegans to analyze the mechanisms that drive apoptosis as well as the mechanisms that determine how cells ensure apoptosis happens when and where it should. Unlike humans and other mammals, which depend on dozens of proteins to control apoptosis, these worms use just a few. And when things go awry, it’s easy to tell: When there’s not enough apoptosis, researchers can see that there are too many cells inside the worms’ translucent bodies. And when there’s too much, the worms lack certain biological functions or, in more extreme cases, can’t reproduce or die during embryonic development.

Work in the Horvitz lab defined the roles of many of the genes and proteins that control apoptosis in worms. These regulators proved to have counterparts in human cells, and for that reason studies of worms have helped reveal how human cells govern cell death and pointed toward potential targets for treating disease.

A protein’s dual role

Three of C. elegans’ primary regulators of apoptosis actively promote cell death, whereas just one, CED-9, reins in the apoptosis-promoting proteins to keep cells alive. As early as the 1990s, however, Horvitz and colleagues recognized that CED-9 was not exclusively a protector of cells. Their experiments indicated that the protector protein also plays a role in promoting cell death. But while researchers thought they knew how CED-9 protected against apoptosis, its pro-apoptotic role was more puzzling.

CED-9’s dual role means that mutations in the gene that encode it can impact apoptosis in multiple ways. Most ced-9 mutations interfere with the protein’s ability to protect against cell death and result in excess cell death. Conversely, mutations that abnormally activate ced-9 cause too little cell death, just like mutations that inactivate any of the three killer genes.

An atypical ced-9 mutation, identified by Reddien when he was a PhD student in Horvitz’s lab, hinted at how CED-9 promotes cell death. That mutation altered the part of the CED-9 protein that interacts with the protein CED-4, which is proapoptotic. Since the mutation specifically leads to a reduction in apoptosis, this suggested that CED-9 might need to interact with CED-4 to promote cell death.

The idea was particularly intriguing because researchers had long thought that CED-9’s interaction with CED-4 had exactly the opposite effect: In the canonical model, CED-9 anchors CED-4 to cells’ mitochondria, sequestering the CED-4 killer protein and preventing it from associating with and activating another key killer, the CED-3 protein —thereby preventing apoptosis.

To test the hypothesis that CED-9’s interactions with the killer CED-4 protein enhance apoptosis, the team needed more evidence. So graduate student Nolan Tucker used CRISPR gene editing tools to create more worms with mutations in CED-9, each one targeting a different spot in the CED-4-binding region. Then he examined the worms. “What I saw with this particular class of mutations was extra cells and viability,” he says—clear signs that the altered CED-9 was still protecting against cell death, but could no longer promote it. “Those observations strongly supported the hypothesis that the ability to bind CED-4 is needed for the pro-apoptotic function of CED-9,” Tucker explains. Their observations also suggested that, contrary to earlier thinking, CED-9 doesn’t need to bind with CED-4 to protect against apoptosis.

When he looked inside the cells of the mutant worms, Tucker found additional evidence that these mutations prevented CED-9’s ability to interact with CED-4. When both CED-9 and CED-4 are intact, CED-4 appears associated with cells’ mitochondria. But in the presence of these mutations, CED-4 was instead at the edge of the cell nucleus. CED-9’s ability to bind CED-4 to mitochondria appeared to be necessary to promote apoptosis, not to protect against it.

Looking ahead

While the team’s findings begin to explain a long-unanswered question about one of the primary regulators of apoptosis, they raise new ones, as well. “I think that this main pathway of apoptosis has been seen by a lot of people as more or less settled science. Our findings should change that view,” Tucker says.

The researchers see important parallels between their findings from this study of worms and what’s known about cell death pathways in mammals. The mammalian counterpart to CED-9 is a protein called BCL-2, mutations in which can lead to cancer.  BCL-2, like CED-9, can both promote and protect against apoptosis. As with CED-9, the pro-apoptotic function of BCL-2 has been mysterious. In mammals, too, mitochondria play a key role in activating apoptosis. The Horvitz lab’s discovery opens opportunities to better understand how apoptosis is regulated not only in worms but also in humans, and how dysregulation of apoptosis in humans can lead to such disorders as cancer, autoimmune disease and neurodegeneration.

An elegant switch regulates production of protein variants during cell division

Cells make variants of thousands of proteins. These variants are not produced indiscriminately, but rather through precise regulatory mechanisms that can meet rapidly changing needs of the cell according to new research from the Cheeseman Lab.

Greta Friar | Whitehead Institute
October 18, 2024

Our cells contain thousands of proteins that have gone largely undetected and unstudied until recent years: these are variants of known proteins, which cells can make when their protein-building machinery interacts differently with the same stretch of genetic code. These protein variants have typically been overlooked as occasional accidents of gene expression, but researchers including Whitehead Institute Member Iain Cheeseman are discovering that they are actually abundant and can play important roles in cell functions. Researchers in Cheeseman’s lab are studying individual protein variants to learn more about them and their roles in health and disease, but they also wanted to understand broader patterns of protein variant production: how do cells control when to make one variant of a protein versus another, and what are the consequences of such switches?

Cheeseman, who is also a professor of biology at the Massachusetts Institute of Technology, and graduate student in his lab Jimmy Ly have now identified how cells switch to a different pattern of protein variant production during mitosis, or cell division. In research published in the journal Nature on October 23, they show that this broad regulatory switch helps cells survive paused cell divisions that can sometimes occur in healthy humans or be triggered by certain chemotherapy treatments. The work confirms that cells make variants of thousands of proteins, and also demonstrates that cells do not do so indiscriminately. Rather, cells use precise regulatory mechanisms to switch between different patterns of protein variant production, in order to rapidly tailor the proteins available to fit the changing needs of the cell.

A plethora of hidden proteins

Hw can our cells contain unknown proteins? In high school biology classes, students learn the rule that each gene codes for exactly one protein, such that if you know an organism’s genetic code, you should know every protein it can make. In fact, there are instead many genes that code for multiple proteins. For a protein to be made, first the genetic code for it is copied from DNA into a messenger RNA (mRNA). Then, a ribosome, the cellular machine that follows the instructions in genetic code to build a protein, locates the coding sequence within the mRNA by scanning for the start codon, a sequence of the three bases A, U, and G – bases are the chemical building blocks of RNA, abbreviated as A, U, C, and G. The ribosome recognizes the AUG start codon as the place to begin following instructions, and builds a protein based on the genetic sequence from there through to another trio of bases called a stop codon. However, one way that different versions of a protein can be produced is that a ribosome may begin reading the instructions from multiple different starting points.

Sometimes, a ribosome may miss the first AUG start codon and skip ahead to another AUG somewhere in the middle of the gene’s code, creating a truncated version of the protein. Sometimes, a ribosome may treat a similar trio of bases, such as CUG or GUG, as a start codon. This can cause it to begin earlier, creating a protein based on an extended genetic sequence. These possibilities mean that cells contain thousands more different proteins, or variants of proteins, than are represented by the dogma of one gene, one protein.

In order to understand protein variant production, the researchers—in collaboration with researchers from Whitehead Institute Member David Bartel’s lab–used a method that let them carefully track ribosomes to compare which start sites ribosomes tended to use. They looked at start site selection during mitosis versus during the rest of the cell cycle and found that a dramatic shift in use occurred for thousands of start sites. Specifically, the researchers found that during mitosis, ribosome scanning becomes more stringent. The ribosome will only begin making proteins at AUG sequences, and even then, only at AUGs that have preferable sequences of bases surrounding them—known as a strong Kozak context. This increased selectivity does not always lead to the familiar version of the protein being made during mitosis; sometimes the first AUG start codon has a weak Kozak context, so a truncated protein gets made from an AUG start codon with a stronger Kozak context that lies within the gene.

“Coming into this project, we knew very little about protein production during mitosis—for a long time, people didn’t think much protein production happened in mitosis at all,” Ly says. “It was satisfying to show not only that it is occurring, but that there’s a shift in which proteins are being made—and that this shift is important for cellular viability.”

How cells switch between protein variant programs

The researchers next identified how the switch to increased stringency is initiated during mitosis. They discovered that the key player is a protein called eIF1, which is one of many partners that can pair with ribosomes to help them select their start site. In particular, increased eIF1 pairing with ribosomes causes the ribosomes to be more stringent in their start codon selection, inhibiting the usage of non-AUG initiation sites or sites with weak Kozak contexts.

During mitosis, ribosome pairing with eIF1 increases sharply, leading to the shift in stringency. This change in pairing rate during mitosis puzzled the researchers: ribosomes and their partners, including eIF1, all typically reside together in the main body of the cell—where ribosomes make proteins—so they should be able to pair freely at any time. The researchers looked for other molecules in the same location that could be altering how ribosomes and eIF1 interact during different parts of the cell cycle, but they couldn’t find anything. Eventually, the researchers realized that the answer to the puzzle lay in a separate location: the nucleus.

They found that cells maintain a large pool of eIF1 inside of the nucleus, locked away from the ribosomes. Then, during cell division, the wall of the nucleus dissolves, mixing its contents with the rest of the cell. This is necessary for the dividing cell to divvy up its DNA, but it also releases the pool of eIF1 to pair with ribosomes, increasing stringency. At the end of mitosis, the nucleus reforms and eIF1 is re-incorporated into the nucleus of each of the two daughter cells, and the cells return to a less stringent program.

“The explanation for increased interaction between eIF1 and ribosomes during mitosis had really stumped us, and so when I saw eIF1 localizing to the nucleus, that was a really exciting ‘aha’ moment,” Ly says. “Discovering this mechanism of nuclear release during mitosis was unexpected, and it’s interesting to think about how else cells might be using it.”

Consequences of increased stringency for the cell

Once the researchers understood the how, they then wanted to understand the why? What they discovered is that when cells have no nuclear pool of eIF1, and so no change in stringency during mitosis, they are more likely to die during mitosis. In particular, these cells fare poorly during mitotic arrest, a state in which cells get stuck in mitosis for hours or even days–much longer than typical mitosis. Arrest occurs when cells detect a possible cell division error and so halt their division until the error is corrected or the cell dies.

One effect of increased stringency during mitosis is related to mitochondria, which are required for energy production in many cell types and are therefore required for maintaining viability. Cells stuck in mitotic arrest need energy to keep them going through this unexpected delay. The researchers found that increased stringency during mitosis led to an increase in the production of important mitochondrial proteins, boosting the cells’ energy supply to get them through arrest.

Increased stringency also gives cells the tools they need to escape arrest, even if they haven’t fixed the error that caused them to pause division. In a Nature paper in 2023, Cheeseman and then-postdoc in his lab Mary-Jane Tsang showed that when cells build up enough of the truncated version of a protein called CDC20, they can escape arrest. Ly’s work adds to this story by showing that the nuclear release of eIF1 increases stringency, leading to more production of truncated CDC20 during mitosis, which explains how cells build up enough of this protein variant during mitosis to trigger their escape. These findings may have important potential implications for some cancer chemotherapy strategies.

Some chemotherapies work by trapping cancer cells in mitotic arrest until they die. Cheeseman, Tsang, and Ly’s work collectively shows that when cancer cells lack sufficient truncated CDC20—as can occur in the absence of nuclear eIF1—the cells cannot escape arrest and so are killed off by these chemotherapies at higher rates. These results could be used to improve the efficacy of antimitotic chemotherapy drugs.

The switch in protein variant production that the researchers found affects thousands of proteins. These newly identified protein variants serve as a foundation for many future projects in the lab.

As the researchers continue to examine the consequences of this switch to stringency during mitosis, they are also searching for other cases in which cells regulate protein variant production outside of mitosis. For example, the researchers are interested in how this switch in stringency affects fertility; immature egg cells spend a long time in a form of arrested cell division without an intact nucleus, and Ly observed eIF1 in the nucleus of the immature female eggs.

“Cells have axes of control that they use to quickly make broad changes in gene expression,” Cheeseman says. “Several of these are central to controlling cell division—for example, the role of phosphorylation as a regulatory switch in mitosis has been well studied. Our work identifies another axis of control, and we’re excited to discover more about when and how cells make use of it.”