Research Area: Biochemistry, Biophysics, and Structural Biology

In search of nature’s winning recipe

Graduate student Darren Parker aims to understand the ratio of ingredients that constitutes the optimal cell.

Raleigh McElvery
May 31, 2019

Fifth-year graduate student Darren Parker is as much a baker as he is a biologist — at least metaphorically speaking. He’s on a mission to understand the ratio of ingredients required to concoct nature’s winning recipe for the optimal cell. Researchers have a solid understanding of which components are essential for cellular function, but they have yet to determine whether it’s critical for cells to generate exactly the right amount of protein.

“In that way, my graduate work is actually pretty simple,” Parker says. “I just want to know if changing the amounts of a specific ingredient has an effect on the overall product.”

The oldest of four brothers, Parker grew up in a suburb just outside of Chicago. When he enrolled in the University of Illinois Urbana-Champaign for his undergraduate studies in 2009, he was considering a major in environmental science. “My high school biology classes were mostly rote memorization,” he explains, “so the molecular aspects just didn’t resonate with me. I was more interested in studying life on a larger scale.”

After his first year, he entered the Integrated Biology program, which essentially “encompassed all biology that wasn’t molecular biology.” He was still required to take an introductory molecular biology course, though, as part of the major. But this time around, something clicked.

He remembers performing his first genetic knockdown experiment, decreasing the level of dopamine receptors in roundworms and witnessing the behavioral ramifications in real time. “I finally had a handle on the molecular concepts enough to really get what was going on,” he says.

He attributed his newfound appreciation for the basic mechanisms underlying life to his fortified chemistry skills. At the beginning of his third year, he officially declared a biochemistry major, and joined a lab in the Department of Chemistry studying nucleic acid enzymes.

Parker’s job was to sift through trillions of short DNA strands, selecting only those that could act like enzymes and cut RNA. He would then home in on the nucleotide sequences within those strands that were best suited to carry out the reaction. After a year-and-a-half, he’d successfully identified a few DNA sequences that could cut RNA molecules with a distinct chemistry. After this point he was excited to try “studying life” as opposed to synthetic reactions.

Mid-way through his fourth year, he joined a biology lab in the College of Medicine probing alternative splicing in liver and heart development. It was a new group with only a few members, and Parker had more experience as an undergraduate than some of the first-year graduate students, so he hit the ground running. His last-minute switch to biochemistry meant he had five years of studies instead of the usual four — totaling six full semesters (and several summers) in lab.

After identifying a key splicing protein required for the liver to fully mature in mice and humans, Parker became even more fascinated by molecular biology and determined to pursue a career in science with bigger picture applications.

At the urging of his advisor, Parker sent in his graduate school applications. He was primarily interested in microbiology and infectious disease research — although he had no prior experience working in bacteria, only a longstanding interest in the intersection of science and society. He ultimately chose MIT Biology because of the breadth of labs. He could join a microbiology lab, or pursue an entirely different path, all within the same department. Gene-Wei Li’s lab seemed like “the perfect mix.”

“Gene was asking questions in molecular biology from the unique perspective of a physicist, looking at biological questions in a way I had never even considered before,” he says. “Gene had also just joined the department and wasn’t tied to a specific field or model organism yet, so I had the chance to build my own projects from the bottom up; I wasn’t just slotting in somewhere.”

Best of all, the Li lab was all about drilling down into to the mechanics of protein production in order to understand the cell as a whole — the bigger picture perspective that Parker was longing for.

Parker began by exploring ways to modify high-throughput RNA sequencing. He aimed to make this popular method cheaper and more scalable, in hopes of knocking out many individual genes in E. coli to test the genome-wide effects. He then pivoted his project and applied his new technique to study the effects of reducing essential genes in B. subtilis, another model bacterium. The family of enzymes that was the most interesting to him from these experiments were the aminoacyl-tRNA synthetases.

tRNAs, or transfer RNAs, carry amino acids to the ribosome so that the cell can produce proteins. This process requires the help of enzymes — tRNA synthetases — to “charge” the tRNAs with an amino acid. Only then can the ribosome transfer the amino acid from the tRNAs to the growing chain of amino acids that eventually forms the protein. Like an inquisitive baker, Parker wanted to know what would happen if he added more or less tRNA synthetase to the recipe of a bacterial cell.

His results would make Goldilocks proud. Over the past few years, he’s shown that too much or too little tRNA synthetase prevents the cell from growing at a normal rate. The amount must be just right.

“It turns out that what’s most important to the cell is maintaining the ratios of those very conserved ingredients,” Parker says. “The cell will actively use less of those ingredients if the synthetase is limiting, and this leads to a much slower growing cell. Adding too much tRNA synthetase is just a waste because the cell already has as much as it needs to sustain translation.”

This same family of tRNA synthetase proteins, he adds, are also implicated in some neurological diseases in humans, which gives him further impetus to study them.

At this point, Parker has taste-tested his fair share of biological areas, and he’s found his niche. “It was a long process,” he says. “That was probably best, though, because it gave me more time to explore.”

Once he graduates, he plans to go into industry, perhaps continuing to tweak the list of ingredients in order to engineer cells to do new things.

“The next time you’re in the kitchen and you want to add more or less of your favorite ingredient,” he urges, “just think about how the cell might feel if you did so with your favorite gene.”

Photo credit: Raleigh McElvery
Posted 5.30.19
The fluid that feeds tumor cells

The substance that bathes tumors in the body is quite different from the medium used to grow cancer cells in the lab, biologists report.

Anne Trafton | MIT News Office
April 16, 2019

Before being tested in animals or humans, most cancer drugs are evaluated in tumor cells grown in a lab dish. However, in recent years, there has been a growing realization that the environment in which these cells are grown does not accurately mimic the natural environment of a tumor, and that this discrepancy could produce inaccurate results.

In a new study, MIT biologists analyzed the composition of the interstitial fluid that normally surrounds pancreatic tumors, and found that its nutrient composition is different from that of the culture medium normally used to grow cancer cells. It also differs from blood, which feeds the interstitial fluid and removes waste products.

The findings suggest that growing cancer cells in a culture medium more similar to this fluid could help researchers better predict how experimental drugs will affect cancer cells, says Matthew Vander Heiden, an associate professor of biology at MIT and a member of the Koch Institute for Integrative Cancer Research.

“It’s kind of an obvious statement that the tumor environment is important, but I think in cancer research the pendulum had swung so far toward genes, people tended to forget that,” says Vander Heiden, one of the senior authors of the study.

Alex Muir, a former Koch Institute postdoc who is now an assistant professor at the University of Chicago, is also a senior author of the paper, which appears in the April 16 edition of the journal eLife. The lead author of the study is Mark Sullivan, an MIT graduate student.

Environment matters

Scientists have long known that cancer cells metabolize nutrients differently than most other cells. This alternative strategy helps them to generate the building blocks they need to continue growing and dividing, forming new cancer cells. In recent years, scientists have sought to develop drugs that interfere with these metabolic processes, and one such drug was approved to treat leukemia in 2017.

An important step in developing such drugs is to test them in cancer cells grown in a lab dish. The growth medium typically used to grow these cells includes carbon sources (such as glucose), nitrogen, and other nutrients. However, in the past few years, Vander Heiden’s lab has found that cancer cells grown in this medium respond differently to drugs than they do in mouse models of cancer.

David Sabatini, a member of the Whitehead Institute and professor of biology at MIT, has also found that drugs affect cancer cells differently if they are grown in a medium that resembles the nutrient composition of human plasma, instead of the traditional growth medium.

“That work, and similar results from a couple of other groups around the world, suggested that environment matters a lot,” Vander Heiden says. “It really was a wake up call for us that to really know how to find the dependencies of cancer, we have to get the environment right.”

To that end, the MIT team decided to investigate the composition of interstitial fluid, which bathes the tissue and carries nutrients that diffuse from blood flowing through the capillaries. Its composition is not identical to that of blood, and in tumors, it can be very different because tumors often have poor connections to the blood supply.

The researchers chose to focus on pancreatic cancer in part because it is known to be particularly nutrient-deprived. After isolating interstitial fluid from pancreatic tumors in mice, the researchers used mass spectrometry to measure the concentrations of more than 100 different nutrients, and discovered that the composition of the interstitial fluid is different from that of blood (and from that of the culture medium normally used to grow cells). Several of the nutrients that the researchers found to be depleted in tumor interstitial fluid are amino acids that are important for immune cell function, including arginine, tryptophan, and cystine.

Not all nutrients were depleted in the interstitial fluid — some were more plentiful, including the amino acids glycine and glutamate, which are known to be produced by some cancer cells.

Location, location, location

The researchers also compared tumors growing in the pancreas and the lungs and found that the composition of the interstitial fluid can vary based on tumors’ location in the body and at the site where the tumor originated. They also found slight differences between the fluid surrounding tumors that grew in the same location but had different genetic makeup; however, the genetic factors tested did not have as big an impact as the tumor location.

“That probably says that what determines what nutrients are in the environment is heavily driven by interactions between cancer cells and noncancer cells within the tumor,” Vander Heiden says.

Scientists have previously discovered that those noncancer cells, including supportive stromal cells and immune cells, can be recruited by cancer cells to help remake the environment around the tumor to promote cancer survival and spread.

Vander Heiden’s lab and other research groups are now working on developing a culture medium that would more closely mimic the composition of tumor interstitial fluid, so they can explore whether tumor cells grown in this environment could be used to generate more accurate predictions of how cancer drugs will affect cells in the body.

The research was funded by the National Institutes of Health, the Lustgarten Foundation, the MIT Center for Precision Cancer Medicine, Stand Up to Cancer, the Howard Hughes Medical Institute, and the Ludwig Center at MIT.

A supportive role for planarians’ multifaceted muscle
Greta Friar | Whitehead Institute
April 5, 2019

CAMBRIDGE, MA  — Planarians are flatworms best known for their incredible ability to regenerate all their body parts: chop a planarian in two and soon you will have two perfectly formed planarians. As Whitehead Institute Member Peter Reddien, also a professor of biology at Massachusetts Institute of Technology and an investigator with the Howard Hughes Medical Institute, has investigated planarians over the years, he has become increasingly fascinated with the functions of their muscle. Not only do planarians use muscle to move, but Reddien’s research group previously discovered they rely on muscle tissue to provide a full body map with instructions that helps guide stem cells to the right locations during both regeneration and normal turnover of cells. Muscle tissue does this by secreting positional signals that help cells identify where they are — and where they should be.

New research from Reddien and graduate student Lauren Cote shows that muscle serves yet another crucial function in planarians. In a paper published in Nature Communications on April 8, they show that muscle operates as the planarian’s connective tissue, providing basic architectural support for the body. Connective tissue functions in large part by secreting molecules that make up the extracellular matrix (ECM), a network of molecules outside of the body’s cells that provides tissues with, among other things, scaffolding, protection, separation of tissues, and a means of inter-tissue connection and communication. In vertebrates, including humans, connective tissue is a distinct tissue type containing dedicated cells such as fibroblasts that secrete most of the animal’s ECM proteins. Reddien and Cote found no such fibroblast-like cell type in planarians; instead, multipurpose muscle does it all.

The researchers began to suspect that planarian muscle might function as connective tissue when they discovered that the gene encoding a major type of ECM molecule, fibrous protein collagen, was expressed only in muscle. The researchers then catalogued the total collection of proteins found in the planarian’s ECM, called the matrisome, and tracked where the genes that code for those proteins were expressed. They identified nineteen collagen genes, and all nineteen were highly specific to muscle. The vast majority of other ECM genes followed suit.

To further test muscle’s role as connective tissue, the researchers silenced the gene hemicentin-1, which produces another ECM molecule expressed specifically in muscle. They found that when the gene was not expressed, the planarian’s inner tissues did not remain properly separated from its outer skin. In other words, a muscle-specific gene is necessary in the planarians they studied for the core connective tissue task of keeping tissues discrete.

Although it might seem unusual that planarians would use muscle tissue for both ECM secretion and body pattern maintenance, Reddien and Cote say the combination makes a certain sense.

“To establish a map of the body, muscle secretes positional signals, and in its role as connective tissue it is simultaneously creating the extracellular environment the signals travel through,” Reddien says.

Cote agrees: “Producing the body’s physical architectural support and its biochemical architectural blueprint seem to go hand in hand.”

One possibility raised by this synchronicity is that a link between connective tissue and harboring positional information exists broadly across animal species. Studies elsewhere have found some positional role or positional memory in connective tissues in several species, including axolotls, vertebrates capable of limb regeneration. Based on these observations, Reddien says, it would be interesting to consider the positional role that connective tissue cells, like fibroblasts, might play in humans and might have in instructing regeneration broadly.

 

Written by Greta Friar

 

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Peter Reddien’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also a Howard Hughes Medical Institute Investigator and a professor of biology at Massachusetts Institute of Technology. The authors also acknowledge the Eleanor Schwartz Charitable Foundation for support.

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Full citation:

“Muscle functions as a connective tissue and source of extracellular matrix in planarians”

Nature Communications, online April 8, 2019. DOI: 10.1038/s41467-019-09539-6

Lauren E. Cote, Eric Simental, and Peter W. Reddien.

Scaffolding the nursery of pollen development
Nicole Giese Rura | Whitehead Institute
April 2, 2019

Cambridge, MA — Increased temperatures and decreased precipitation associated with climate change could threaten the world’s crops. Seed and pollen production in particular are vulnerable to shifts in temperature or rainfall. For example, in heat- or drought-stressed wheat and rice, the tissue responsible for nourishing pollen, called the tapetum, is compromised, causing the plants to not generate pollen. Without pollen, these staples are unable to bear the grains that billions of people rely on for food. In research described this week in the journal Plant Cell, Whitehead Institute Member Jing-Ke Weng and his lab have identified the components of a critical scaffold system that supports the tapetum. With a better understanding of the tapetum, scientists may be able to adapt plants to produce pollen even in hot, arid conditions.

Within a flower bud, pollen-filled anthers perch atop stalk-like filaments. Lining the anther’s inner chamber is a tissue called the tapetum, which nurtures the developing pollen. To better understand pollen and anther formation, Joseph Jacobowitz, a graduate student in Weng’s lab and first author of the Plant Cell paper, analyzed genes active in the anther during early flower development in the Arabidopsis plant. Two practically unknown genes stood out because they likely contribute to pollen maturation: PRX9 and PRX40. After further investigation, Jacobowitz determined that the two genes encode enzymes that work in conjunction with another type of protein called extensin and together they form the supportive walls that act like a scaffold in the tapetum.

Weng, who is also an assistant professor of biology at Massachusetts Institute of Technology, likens extensins to bricks in a wall and the PRX9 and PRX40 proteins to the mortar. Pushing against a wall can easily compromise its structure unless mortar bonds the bricks together. The same seems to be true with extensins and PRX9 and PRX40. The extensins and PRX9/PRX40 wall in the tapetum remained intact until Jacobowitz genetically “knocked out” the mortar genes. With the mortar gone, the scaffolding loses its integrity, and the tapetum collapses into the space where the pollen develops, either crushing or starving it. The result appears similar to what occurs in the tapetum of stressed wheat and rice plants, and the final effects are similar as well: Both the stressed crops and Arabidopsis lacking PRX9 and PRX40 are male sterile and do not produce pollen.

After further investigation, Jacobowitz and colleagues determined that the PRX9 and PRX40 genes are closely related and first appeared at pivotal moments in plant history. PRX40 is highly conserved among land plants and originated about 470 million years ago, when plants first emerged onto land from the seas and rivers. PRX9 seems to have evolved from PRX40 as a redundant backup when flowering plants diverged from nonflowering plants.

Pollen creation is a delicate process that plants have evolved over millions of years. Insights such as these into how plants maintain the integrity of their reproductive system are invaluable toward understanding how we might be able to generate crops capable of withstanding environmental stresses like heat and drought that could threaten our food supply.

This work was supported by Pew Scholars Program in the Biomedical Sciences (27345), the Searle Scholars Program (15-SSP-162), and the National Science Foundation (CHE-1709616 and 1122374).

Written by Nicole Giese Rura

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Jing-Ke Weng’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also an assistant professor of biology at Massachusetts Institute of Technology.

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Citation:

“PRX9 and PRX40 are extensin peroxidases essential for maintaining tapetum and microspore cell wall integrity during Arabidopsis anther development”

Plant Cell, online March 18, 2019, DOI: https://doi.org/10.1105/tpc.18.00907

Joseph R. Jacobowitz, William C. Doyle, and Jing-Ke Weng.

Predicting sequence from structure

Researchers have devised a faster, more efficient way to design custom peptides and perturb protein-protein interactions.

Raleigh McElvery | Department of Biology
February 15, 2019

One way to probe intricate biological systems is to block their components from interacting and see what happens. This method allows researchers to better understand cellular processes and functions, augmenting everyday laboratory experiments, diagnostic assays, and therapeutic interventions. As a result, reagents that impede interactions between proteins are in high demand. But before scientists can rapidly generate their own custom molecules capable of doing so, they must first parse the complicated relationship between sequence and structure.

Small molecules can enter cells easily, but the interface where two proteins bind to one another is often too large or lacks the tiny cavities required for these molecules to target. Antibodies and nanobodies bind to longer stretches of protein, which makes them better suited to hinder protein-protein interactions, but their large size and complex structure render them difficult to deliver and unstable in the cytoplasm. By contrast, short stretches of amino acids, known as peptides, are large enough to bind long stretches of protein while still being small enough to enter cells.

The Keating lab at the MIT Department of Biology is hard at work developing ways to quickly design peptides that can disrupt protein-protein interactions involving Bcl-2 proteins, which promote cancer growth. Their most recent approach utilizes a computer program called dTERMen, developed by Keating lab alumnus, Gevorg Grigoryan PhD ’07, currently an associate professor of computer science and adjunct associate professor of biological sciences and chemistry at Dartmouth College. Researchers simply feed the program their desired structures, and it spits out amino acid sequences for peptides capable of disrupting specific protein-protein interactions.

“It’s such a simple approach to use,” says Keating, an MIT professor of biology and senior author on the study. “In theory, you could put in any structure and solve for a sequence. In our study, the program came up with new sequence combinations that aren’t like anything found in nature — it deduced a completely unique way to solve the problem. It’s exciting to be uncovering new territories of the sequence universe.”

Former postdoc Vincent Frappier and Justin Jenson PhD ’18 are co-first authors on the study, which appears in the latest issue of Structure.

Same problem, different approach

Jenson, for his part, has tackled the challenge of designing peptides that bind to Bcl-2 proteins using three distinct approaches. The dTERMen-based method, he says, is by far the most efficient and general one he’s tried yet.

Standard approaches for discovering peptide inhibitors often involve modeling entire molecules down to the physics and chemistry behind individual atoms and their forces. Other methods require time-consuming screens for the best binding candidates. In both cases, the process is arduous and the success rate is low.

dTERMen, by contrast, necessitates neither physics nor experimental screening, and leverages common units of known protein structures, like alpha helices and beta strands — called tertiary structural motifs or “TERMs” — which are compiled in collections like the Protein Data Bank. dTERMen extracts these structural elements from the data bank and uses them to calculate which amino acid sequences can adopt a structure capable of binding to and interrupting specific protein-protein interactions. It takes a single day to build the model, and mere seconds to evaluate a thousand sequences or design a new peptide.

“dTERMen allows us to find sequences that are likely to have the binding properties we’re looking for, in a robust, efficient, and general manner with a high rate of success,” Jenson says. “Past approaches have taken years. But using dTERMen, we went from structures to validated designs in a matter of weeks.”

Of the 17 peptides they built using the designed sequences, 15 bound with native-like affinity, disrupting Bcl-2 protein-protein interactions that are notoriously difficult to target. In some cases, their designs were surprisingly selective and bound to a single Bcl-2 family member over the others. The designed sequences deviated from known sequences found in nature, which greatly increases the number of possible peptides.

“This method permits a certain level of flexibility,” Frappier says. “dTERMen is more robust to structural change, which allows us to explore new types of structures and diversify our portfolio of potential binding candidates.”

Probing the sequence universe

Given the therapeutic benefits of inhibiting Bcl-2 function and slowing tumor growth, the Keating lab has already begun extending their design calculations to other members of the Bcl-2 family. They intend to eventually develop new proteins that adopt structures that have never been seen before.

“We have now seen enough examples of various local protein structures that computational models of sequence-structure relationships can be inferred directly from structural data, rather than having to be rediscovered each time from atomistic interaction principles,” says Grigoryan, dTERMen’s creator. “It’s immensely exciting that such structure-based inference works and is accurate enough to enable robust protein design. It provides a fundamentally different tool to help tackle the key problems of structural biology — from protein design to structure prediction.”

Frappier hopes one day to be able to screen the entire human proteome computationally, using methods like dTERMen to generate candidate binding peptides. Jenson suggests that using dTERMen in combination with more traditional approaches to sequence redesign could amplify an already powerful tool, empowering researchers to produce these targeted peptides. Ideally, he says, one day developing peptides that bind and inhibit your favorite protein could be as easy as running a computer program, or as routine as designing a DNA primer.

According to Keating, although that time is still in the future, “our study is the first step towards demonstrating this capacity on a problem of modest scope.”

This research was funded the National Institute of General Medical Sciences, National Science Foundation, Koch Institute for Integrative Cancer Research, Natural Sciences and Engineering Research Council of Canada, and Fonds de Recherche du Québec.

Why too much DNA repair can injure tissue

Overactive repair system promotes cell death following DNA damage by certain toxins, study shows.

Anne Trafton | MIT News Office
February 14, 2019

DNA-repair enzymes help cells survive damage to their genomes, which arises as a normal byproduct of cell activity and can also be caused by environmental toxins. However, in certain situations, DNA repair can become harmful to cells, provoking an inflammatory response that produces severe tissue damage.

MIT Professor Leona Samson has now determined that inflammation is a key component of the way this damage occurs in photoreceptor cells in the retinas of mice. About 10 years ago, she and her colleagues discovered that overactive initiation of DNA-repair systems can lead to retinal damage and blindness in mice. The key enzyme in this process, known as Aag glycosylase, can also cause harm in other tissues when it becomes hyperactive.

“It’s another case where despite the fact that inflammation is there to protect you, in some circumstances it can actually be harmful, when it’s overactive,” says Samson, a professor emerita of biology and biological engineering and the senior author of the study.

Aag glycosylase helps to repair DNA damage caused by a class of drugs known as alkylating agents, which are commonly used as chemotherapy drugs and are also found in pollutants such as tobacco smoke and fuel exhaust. Retinal damage from these drugs has not been seen in human patients, but alkylating agents may produce similar damage in other human tissues, Samson says. The new study, which reveals how Aag overactivity leads to cell death, suggest possible targets for drugs that could prevent such damage.

Mariacarmela Allocca, a former MIT postdoc, is the lead author of the study, which appears in the Feb. 12 issue of Science Signaling. MIT technical assistant Joshua Corrigan, former postdoc Aprotim Mazumder, and former technical assistant Kimberly Fake are also authors of the paper.

A vicious cycle

In a 2009 study, Samson and her colleagues found that a relatively low level of exposure to an alkylating agent led to very high rates of retinal damage in mice. Alkylating agents produce specific types of DNA damage, and Aag glycosylase normally initiates repair of such damage. However, in certain types of cells that have higher levels of Aag, such as mouse photoreceptors, the enzyme’s overactivity sets off a chain of events that eventually leads to cell death.

In the new study, the researchers wanted to find exactly out how this happens. They knew that Aag was overactive in the affected cells, but they didn’t know exactly how it was leading to cell death or what type of cell death was occurring. The researchers initially suspected it was apoptosis, a type of programmed cell death in which a dying cell is gradually broken down and absorbed by other cells.

However, they soon found evidence that another type of cell death called necrosis accounts for most of the damage. When Aag begins trying to repair the DNA damage caused by the alkylating agent, it cuts out so many damaged DNA bases that it hyperactivates an enzyme called PARP, which induces necrosis. During this type of cell death, cells break apart and spill out their contents, which alerts the immune system that something is wrong.

One of the proteins secreted by the dying cells, known as HMGB1, stimulates production of chemicals that attract immune cells called macrophages, which specifically penetrate the photoreceptor layer of the retina. These macrophages produce highly reactive oxygen species — molecules that create more damage and make the environment even more inflammatory. This in turn causes more DNA damage, which is  recognized by Aag.

“That makes the situation worse, because the Aag glycosylase will act on the lesions produced from the inflammation, so you get a vicious cycle, and the DNA repair drives more and more degeneration and necrosis in the photoreceptor layer,” Samson says.

None of this happens in mice that lack Aag or PARP, and it does not occur in other cells of the eye or in most other body tissues.

“It amazes me how segmented this is. The other cells in the retina are not affected at all, and they must experience the same amount of DNA damage. So, one possibility is maybe they don’t express Aag, while the  photoreceptor cells do,” Samson says.

“These molecular studies are exciting, as they have helped define the underlying pathophysiology associated with retinal damage,” says Ben Van Houten, a professor of pharmacology and chemical biology at the University of Pittsburgh, who was not involved in the study. “DNA repair is essential for the faithful inheritance of a cell’s genetic material. However, the very action of some DNA repair enzymes can result in the production of toxic intermediates that exacerbate exposures to genotoxic agents.”

Varying effects

The researchers also found that retinal inflammation and necrosis were more severe in male mice than in female mice. They suspect that estrogen, which can interfere with PARP activity, may help to suppress the pathway that leads to inflammation and cell death.

Samson’s lab has previously found that Aag activity can also exacerbate damage to the brain during a stroke, in mice. The same study revealed that Aag activity also worsens inflammation and tissue damage in the liver and kidney following oxygen deprivation. Aag-driven cell death has also been seen in the mouse cerebellum and some pancreatic and bone marrow cells.

The effects of Aag overactivity have been little studied in humans, but there is evidence that healthy individuals have widely varying levels of the enzyme, suggesting that it could have different effects in different people.

“Presumably there are some cell types in the human body that would respond the same way as the mouse photoreceptors,” Samson says. “They may just not be the same set of cells.”

The research was funded by the National Institutes of Health.

Puzzling over Pollen

Graduate student Joe Jacobowitz analyzes new enzymes that could reveal key insights into plant reproduction.

Raleigh McElvery
January 24, 2019

Every morning, fifth-year graduate student Joe Jacobowitz takes the elevator to the seventh floor of the Whitehead Institute, passes the soil bins, “false winter” fridges, and toasty growing chambers, and enters his favorite workspace: the greenhouse. There, among the myriad of tall, stout, grass-like, and blooming plants, he attends to his organism of choice, Arabidopsis thaliana. With four simple, white petals interrupted by protruding, yellow stamens, “it looks like something that would grow in the cracks of a sidewalk,” Jacobowitz says. While you or I might pass by it and not think twice, Jacobowitz and the Weng lab hold that Arabidopsis could reveal key insights into pollen development, in particular which enzymes are critical for plant reproduction.

Jacobowitz became fascinated by enzymes as a biochemistry major at Brandeis University, studying the evolution of a single enzyme found in the deadliest form of malaria. After arriving at MIT Biology for graduate school and joining Jing-Ke Weng’s team at the Whitehead, Jacobowitz shifted his focus from biochemistry and biophysics to plant development. His work investigating the pollen-bearing chamber known as the anther represents just one facet of the Weng lab — which probes the origin and evolution of plant metabolism, as well as the small molecules plants produce to interact with their environments.

Above his lab desk, next to hand-drawn sketches and photos of friends, Jacobowitz has taped intricate microscopy images detailing the many complex stages of anther development. The pollen grains inside this structure contain the plant’s male gametes, which are transferred via wind and passersby to the female part, the pistil, of another flower. In the case of Arabidopsis, a single flower can self-pollinate and reproduce on its own, generating seeds and engendering the next generation. As the pollen grains mature, they become coated in a tough outer layer made of the material sporopollenin. This polymer, Jacobowitz explains, has helped sculpt the terrestrial ecosystem we know today.

Nearly 500 million years ago, the first plants migrated from sea to land, and eventually developed this durable coating to protect their delicate pollen grains from the stresses of living above water, such as UV radiation and desiccation. Today, researchers understand the basic sequence of events required for pollen development, but it’s been historically difficult to identify the genes involved — or even break down the resilient sporopollenin to determine its composition. In December of 2018, Weng lab postdoc Fu-Shuang Li and his team became the first to report the successful degradation of this virtually indestructible material and determine its chemical structure.

“Now that we have a better grasp of what this pollen coating looks like at a molecular level,” says co-author Jacobowitz, “we can improve our understanding of the genes that are already known to produce the pollen wall, and make predictions about new enzymes that also likely contribute.”

Jacobowitz aims to pinpoint which enzymes add certain chemical groups to sporopollenin, as well as the molecular players required for anther development. As he puts it, the general premise of his current project is to “examine genes that no one has looked at before.”

Jacobowitz spent almost a year sifting through online databases to compile a list of enzymes that could potentially play a critical role in anther development. He ordered knockout lines that eliminated each enzyme one at a time, and watched as the plants matured.

At first, nothing happened. Jacobowitz was simply rearing a bunch of normal plants. But then it occurred to him that perhaps nature had built in some redundancy, allowing plants to survive these genetic errors. If one enzyme was incapacitated, another might compensate for the loss and assume its function so development could proceed as usual.

“Even though my screens were pretty unsuccessful at first, I still enjoyed the entire process,” he recalls. “That’s when I started to realize that I really like genetics. There’s always this possibility that you’ll stumble upon a new gene, or a new function of a known gene, that no one ever suspected. That was the opposite of my undergraduate experience in biochemistry, where we drilled down into the intimate details of a single, well-studied enzyme.”With this in mind, Jacobowitz crossed two knockouts together and created a double mutant, simultaneously erasing what he suspected were two relatively similar enzymes. This time, he saw an effect — the walls of the anther began to swell, invading the space containing the pollen and preventing the grains from developing properly. He’d made a sterile plant, indicating that these two enzymes (encoded by the PRX9 and PRX40 genes, respectively) were critical for pollen development

Post-MIT, Jacobowitz is considering pursing a postdoc in genetics. He’s open to studying any organism, so plants aren’t off the table just yet.

“As humans, we rely heavily on plant-based medicines and agricultural products,” he says. “In today’s changing climate, it’s especially important recognize our dependence on plants, and put necessary resources into understanding the basic principles governing their reproductive cycle.” In fact, our own lives could depend on it.

Posted 1.24.19