Research Area: Microbiology

Researchers determine what makes some proteins “slippery” enough to evade destruction
Raleigh McElvery
July 10, 2019

All cells must balance generating new proteins with eliminating excess or damaged ones by way of powerful degradation machines — which, much like wood chippers, chew up proteins and spit them out. But, these proteins are often folded into intricate structures, and must be unfurled before they can be fed into these degradation machines, broken into tiny bits, and ultimately recycled. In bacteria, a molecular motor known as ClpX must grip the end of the ill-fated protein and apply force to straighten it. However, until now, researchers weren’t sure precisely how ClpX gripped its target tightly enough to accomplish this task.

There had been evidence to suggest that some amino acids — the chemical building blocks that comprise proteins — are “slippery” and thus more difficult to grip. In a new study published in eLife, researchers at the MIT Department of Biology examined each amino acid’s individual contribution to grip. By parsing the physical basis for this molecular interaction, they hope to better understand how some proteins evade destruction.

“Previous studies had shown that small amino acids were notoriously hard to grip, but no one really understood why,” says Tristan Bell, graduate student and first author on the paper. “It’s like watching a game of tug-of-war and knowing that a person’s hands are important for pulling on the rope, but having no idea what allows the hands to get a good grip on the rope.”

ClpX, he explains, is roughly shaped like donut with loops protruding into the center hole. These loops grip the target protein, jamming it against the surface of ClpX, and unfolding it so it can be threaded through the hole and shredded.

The researchers engineered proteins with tails comprised of various amino acid combinations, and measured how well ClpX could grip them, both in bacteria and in test tubes. They determined that ClpX can only grip between six and eight amino acids at a time, and that only a handful of the 20 possible amino acids could actually be “well-gripped.” When ClpX was able to grasp multiple amino acids simultaneously, its grip strength increased.

“We think that somehow the charge is preventing ClpX from making strong contacts with the target protein, preventing it from achieving a stable grip state,” Bell says.Just like in previous experiments, large amino acids appeared easier to grip than small ones, “similar to the way a knotted rope is easier to grasp than a smooth, slippery one,” Bell says. But, regardless of size, amino acids that carried electric charge seemed to be more slippery.

The team thinks that proteins with slippery tails might have an evolutionary advantage, because they are harder to grip and therefore less likely to be degraded.

Invaders like viruses have been known to insert a slippery sequence into certain proteins to prevent the host cell from destroying them and thus promoting replication. Even healthy cells produce proteins with strategically placed slippery sequences, which allow a portion of the protein to break away from the degradation machinery unscathed. In the bacteria Caulobacter crescentus, this planned breakage actually produces a version of one protein that’s needed for DNA replication.

“Next,” Bell says, “we’re hoping to look across entire proteomes in different organisms to find more proteins that escape destruction.”

“Tristan’s experiments and results reveal some of the molecular determinants of grip in the bacterial degradation machines we study,” says Bob Sauer, the Salvador E. Luria Professor of Biology and senior author on the study. “Many of the rules he discovered apply to related machines that function in all biological organisms, including humans, emphasizing the common evolution of these machines.”

Citation:
“Interactions between a subset of substrate side chains and AAA+ motor pore loops determine grip during protein unfolding”
eLife, online June 28, 2019, DOI: 10.7554/eLife.46808
Tristan A. Bell, Tania A. Baker, and Robert T. Sauer.

Researchers identify important proteins hijacked by pathogens during cell-to-cell spread
Raleigh McElvery
July 9, 2019

Listeria monocytogenes, the food-borne bacterium responsible for listeriosis, can creep from one cell to the next, stealthily evading the immune system. This strategy of cell-to-cell spread allows them to infect many different cell types, and can spur complications like meningitis. Yet the molecular details of this spread remain a mystery.

In a paper recently published in Molecular Biology of the Cell, researchers from the MIT Department of Biology, University of California, Berkeley, and Chan Zuckerberg Biohub are beginning to piece together the elusive means by which Listeria moves from one cell to the next. This mode of transport, the scientists suggest, looks a lot like trans-endocytosis, a process that healthy, uninfected cells use to exchange organelles and various cytoplasmic components. In fact, the two processes are so similar that Listeria may be co-opting the host cell’s trans-endocytosis machinery for its own devices.

Although the particulars of trans-endocytosis are poorly understood, the process permits neighboring cells to exchange materials via membrane-bound compartments called vacuoles, which release their cargo upon reaching their final destination.

Much like trans-endocytosis, cell-to-cell spread relies on vacuoles to ferry Listeria. First, the pathogen commandeers the host cell’s own machinery to assemble a tail of proteins that allows it to rocket around inside the cell and ram against both the membrane of the host and that of the adjacent cell. The resulting protrusion is then somehow engulfed into a double-membrane vacuole, and the bacteria burst through their containment to begin the process anew in the recipient cell.

“There’s been a lot of work looking at Listeria cell-to-cell spread,” says Rebecca Lamason, the Robert A. Swanson (1969) Career Development Assistant Professor in the MIT Department of Biology and senior author on the study. “But we still don’t really understand the molecular mechanisms that allow the bacteria to manipulate the membrane to promote engulfment. Depending on what we uncover, we might also be able to apply that information to better grasp how an uninfected cell regulates trans-endocytosis.”

Lamason and her team anticipated that the same proteins implicated in trans-endocytosis would also be involved in Listeria cell-to-cell spread, which would indicate that the pathogen was appropriating these proteins for its own purposes. The researchers made a list of 115 host genes of interest, and then used an RNAi screen to identify just 22 that are critical for cell-to-cell spread.

They were excited to find that, of those 22 genes, several are also implicated in endocytosis, which suggests Listeria is using a similar strategy. These include genes encoding caveolin proteins that control membrane trafficking and remodeling, as well as another protein called PACSIN2 that interacts with caveolins to regulate protrusion engulfment.

Now that the researchers have pinpointed these key proteins, the next step is to determine how they work together in order to promote cell-to-cell spread — especially since the protrusions created by Listeria are much larger than those required for trans-endocytosis.

“As we drill down even deeper into the molecular mechanisms, it will be interesting to see where trans-endocytosis and cell-to-cell spread differ, and where they are similar,” Lamason says. “Our hope is that investigating the mechanisms of bacterial spread will reveal fundamental insights into host intercellular communication.”

Citation:
“RNAi screen reveals a role for PACSIN2 and caveolins during bacterial cell-to-cell spread”
Molecular Biology of the Cell, online June 26, 2019, DOI: 10.1091/mbc.E19-04-0197
Allen G. Sanderlin, Cassandra Vondrak, Arianna J. Scricco, Indro Fedrigo, Vida Ahyong, and Rebecca L. Lamason

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
Origin story

Junior Leah McKinney practiced kitchen microbiology on her ranch in Nevada before exploring the intricacies of DNA replication initiation in bacteria at MIT Biology.

Raleigh McElvery
February 6, 2019

Leah McKinney grew up on a 50,000-acre cattle ranch in Nevada — vaccinating sheep, roping calves, digging for fossils, and occasionally hauling home old bovine femurs. She saddled horses, treated sick lambs, and helped ewes struggling to give birth. One Christmas, she even asked Santa for a fetal pig. “He delivered,” McKinney, now a junior in Course 7, recalls with a laugh.

When she was 12 years old, she saved up enough birthday money to purchase a microscope. Even though she permanently dyed the kitchen sink a distinct shade of blue while making slides, her parents (who both hold degrees in animal science) didn’t mind. They even let her grow bacteria in the heater closet and tally them on the kitchen counter — all in the name of the elementary school science fair.

“They were always encouraging my weird scientific endeavors,” she says. “I think my love for science, and microbiology specifically, came out of my agricultural upbringing.”

She grew to appreciate basic science because it allowed her to study the fundamental mechanisms behind key biological processes. She arrived at MIT in 2016 determined to major in Biology, and hasn’t wavered in her decision. Although she relishes the subject matter, she initially feared the classes would be tedious and memorization-heavy.

“I was quite happy to learn that’s not the case here,” she says. “MIT Biology values problem-solving over rote memorization, and encourages you to take the information you’ve learned in class and apply it to interesting problems. And that mindset extends from the classroom into the lab.”

One of the things that drew McKinney to MIT was the institute’s Undergraduate Research Opportunities Program (UROP), which allows students to join labs and collaborate with faculty as early as their first year. She recalls that, while other universities touted similar opportunities, MIT placed theirs front and center.

“I’d heard that all you had to do was email a professor and ask to join the lab, but I didn’t believe it — that just seemed way too easy,” McKinney says. “But when I was looking for a UROP, I just emailed my current principal investigator to set up a time to talk, and now I’ve been in his lab for over a year.”

McKinney is part of Department Head Alan Grossman’s lab, which investigates the molecular mechanisms and regulation underlying basic cellular processes in bacteria. The entire group works with the rod-shaped Bacillus subtilis, but some members study horizontal gene transfer while others focus on DNA replication and gene expression. McKinney and her graduate student mentor Mary Anderson are in this second category, examining a protein called DnaA that is required to initiate DNA replication and also modulates the expression of several genes.

In order to successfully grow and reproduce, a bacterium must first replicate its single chromosome before dividing into two identical daughter cells. DnaA is responsible for beginning DNA replication in all bacteria. It binds to the origin of replication on the chromosome, unwinds some of the nearby DNA, and recruits the other proteins needed to copy the chromosome.

This operation is highly regulated to ensure that each daughter cell receives only a single chromosome. B. subtilis controls replication via several proteins, including YabA. When YabA binds to DnaA, it prevents replication from ever getting started.

Since DnaA also serves as a transcription factor — binding to other DNA sequences called promoters to increase or decrease expression of certain genes, including its own gene dnaA — YabA may also impact DnaA’s gene targets. McKinney hopes to eventually determine exactly how.

While McKinney discovers something new about her bacteria each time she conducts a successful experiment, she learns almost as much when her tests go awry. “I’ve had to practice a lot of troubleshooting,” she says, “and that’s not something you can learn in class. But everyone in the lab is incredibly friendly and always willing to answer questions or give advice.”

As a teaching assistant for the lab class 7.02 (Introduction to Experimental Biology and Communication), McKinney had the chance to help other students conduct experiments, answering their questions and grading their lab notebooks. She took 7.02 last spring, but says it’s been enlightening to experience the class through a different lens. She adds: “I definitely understand the material more deeply than I did before.”

In addition to TAing, McKinney teaches an SAT preparatory program run by MIT students. “At first, standing up and talking in front of a 20-person section was rather terrifying, but it’s become so much easier,” she says. “The experience has been really good for me.”

After she graduates, McKinney knows she wants to go to graduate school — likely for microbiology — but beyond that, nothing is concrete. She is sure of one thing, though: joining the Grossman lab was one of the best decisions she’s made at MIT.

She advises all current and prospective students to do a UROP. “Find something you’re really interested in,” she says. “It’s okay not to know a lot coming in; you’re going to learn so much, including topics and techniques you won’t learn in class. And don’t be too disappointed when things don’t work; that’s just part of the process. And when you finally get something to work that you’ve been troubleshooting for a while, the feeling is absolutely amazing.”

Posted 2.5.19
Bacteria promote lung tumor development, study suggests

Antibiotics or anti-inflammatory drugs may help combat lung cancer.

Anne Trafton | MIT News Office
January 31, 2019

MIT cancer biologists have discovered a new mechanism that lung tumors exploit to promote their own survival: These tumors alter bacterial populations within the lung, provoking the immune system to create an inflammatory environment that in turn helps the tumor cells to thrive.

In mice that were genetically programmed to develop lung cancer, those raised in a bacteria-free environment developed much smaller tumors than mice raised under normal conditions, the researchers found. Furthermore, the researchers were able to greatly reduce the number and size of the lung tumors by treating the mice with antibiotics or blocking the immune cells stimulated by the bacteria.

The findings suggest several possible strategies for developing new lung cancer treatments, the researchers say.

“This research directly links bacterial burden in the lung to lung cancer development and opens up multiple potential avenues toward lung cancer interception and treatment,” says Tyler Jacks, director of MIT’s Koch Institute for Integrative Cancer Research and the senior author of the paper.

Chengcheng Jin, a Koch Institute postdoc, is the lead author of the study, which appears in the Jan. 31 online edition of Cell.

Linking bacteria and cancer

Lung cancer, the leading cause of cancer-related deaths, kills more than 1 million people worldwide per year. Up to 70 percent of lung cancer patients also suffer complications from bacterial infections of the lung. In this study, the MIT team wanted to see whether there was any link between the bacterial populations found in the lungs and the development of lung tumors.

To explore this potential link, the researchers studied genetically engineered mice that express the oncogene Kras and lack the tumor suppressor gene p53. These mice usually develop a type of lung cancer called adenocarcinoma within several weeks.

Mice (and humans) typically have many harmless bacteria growing in their lungs. However, the MIT team found that in the mice engineered to develop lung tumors, the bacterial populations in their lungs changed dramatically. The overall population grew significantly, but the number of different bacterial species went down. The researchers are not sure exactly how the lung cancers bring about these changes, but they suspect one possibility is that tumors may obstruct the airway and prevent bacteria from being cleared from the lungs.

This bacterial population expansion induced immune cells called gamma delta T cells to proliferate and begin secreting inflammatory molecules called cytokines. These molecules, especially IL-17 and IL-22, create a progrowth, prosurvival environment for the tumor cells. They also stimulate activation of neutrophils, another kind of immune cell that releases proinflammatory chemicals, further enhancing the favorable environment for the tumors.

“You can think of it as a feed-forward loop that forms a vicious cycle to further promote tumor growth,” Jin says. “The developing tumors hijack existing immune cells in the lungs, using them to their own advantage through a mechanism that’s dependent on local bacteria.”

However, in mice that were born and raised in a germ-free environment, this immune reaction did not occur and the tumors the mice developed were much smaller.

Blocking tumor growth

The researchers found that when they treated the mice with antibiotics either two or seven weeks after the tumors began to grow, the tumors shrank by about 50 percent. The tumors also shrank if the researchers gave the mice drugs that block gamma delta T cells or that block IL-17.

The researchers believe that such drugs may be worth testing in humans, because when they analyzed human lung tumors, they found altered bacterial signals similar to those seen in the mice that developed cancer. The human lung tumor samples also had unusually high numbers of gamma delta T cells.

“If we can come up with ways to selectively block the bacteria that are causing all of these effects, or if we can block the cytokines that activate the gamma delta T cells or neutralize their downstream pathogenic factors, these could all be potential new ways to treat lung cancer,” Jin says.

Many such drugs already exist, and the researchers are testing some of them in their mouse model in hopes of eventually testing them in humans. The researchers are also working on determining which strains of bacteria are elevated in lung tumors, so they can try to find antibiotics that would selectively kill those bacteria.

The research was funded, in part, by a Lung Cancer Concept Award from the Department of Defense, a Cancer Center Support (core) grant from the National Cancer Institute, the Howard Hughes Medical Institute, and a Margaret A. Cunningham Immune Mechanisms in Cancer Research Fellowship Award.

Engineering “capture compounds” to probe cell growth

Researchers develop a method to investigate how bacteria respond to starvation and to identify which proteins bind to what they call the “magic spot” — ppGpp.

Raleigh McElvery | Department of Biology
December 17, 2018

In 1969, scientist Michael Cashel was analyzing the compounds produced by starved bacteria when he noticed two spots appearing on his chromatogram as if by magic. Today, we know one of these “magic spots,” as researchers call them, as guanosine tetraphosphate, or ppGpp for short. We also understand that it is a signaling molecule present in virtually all bacteria, helping tune cell growth and size based on nutrient availability.

And yet, despite decades of study, precisely how ppGpp regulates bacterial growth has remained rather mysterious. Delving further requires a more comprehensive list of the molecules that ppGpp binds to exert its effects.

Now, collaborators from MIT’s departments of Biology and Chemistry have developed a method to do just that, and used their new approach to pinpoint over 50 ppGpp targets in Escherichia coli — roughly half which had not been identified previously. Many of these targets are enzymes required to produce nucleotides, the building blocks of DNA and RNA. During times when the bacteria do not have enough nutrients to grow and divide normally, the researchers propose that ppGpp prevents these enzymes from creating new nucleotides from scratch, helping cells enter a dormant state.

“With small molecules or metabolites like ppGpp, it’s been difficult historically to determine which proteins they bind,” says Michael Laub, a professor of biology, a Howard Hughes Medical Institute investigator, and the senior author of the study. “This has been an intractable problem that’s held the field back for some time, but our new approach allows you to nail down the likely targets in a matter of weeks.”

Postdoc Boyuan Wang is the first author of the study, which appeares in Nature Chemical Biology on Dec. 17.

Since ppGpp was discovered nearly 50 years ago, it has been shown to suppress DNA replication, transcription, translation, and various metabolic pathways. It puts the brakes on cell growth and allows bacteria to persist in the face of starvation, stress, and antibiotics. Its influence over numerous regulatory processes has remained somewhat of a mystery, however — after all, it doesn’t just modulate a single pathway but coordinates multiple operations simultaneously to orchestrate a mass shutdown of the cell.

In order to discern which proteins ppGpp binds to effect such widespread change, the researchers built what they call “capture compounds” that contain ppGpp, allowing them to fish out its targets from bacterial extracts. These compounds included a photoreactive crosslinker that latched tightly onto the proteins of interest in the presence of light, and a biotin handle that helped the scientists pull out the proteins to identify them. Most importantly, they were joined to ppGpp in such a way that they wouldn’t interfere with its ability to bind to its targets. This method is more efficient and accurate compared to more traditional means of distinguishing ppGpp targets, which are far more arduous and lack sensitivity.

“Our approach solves these problems because you’re no longer required to do such labor-intensive protocols in order to identify ppGpp targets — and it works even in bacteria beyond E. coli,” says Wang. “Although ppGpp is common among many bacterial species, it seems to exert its effects through different mechanisms, which complicates things. Our capture compounds provide a way to unravel this diversity, and in short order.”

Although the 56 ppGpp targets Wang identified in his screen control a myriad of cellular processes, he homed in on the enzyme PurF — which initiates the biosynthesis of purine nucleotides bearing adenine and guanine bases, also known as A and G.

When bacteria are stressed or starved, they enter a dormant state to survive. But simply curbing translation and transcription is not enough; nucleotides are still being generated and will build up if their synthesis is not put on pause. Cells can build nucleotides in one of two ways: either by salvaging existing materials or starting completely from scratch. PurF kicks off the first step in the latter process leading to the A and G nucleotides. However, when ppGpp binds to PurF, it causes the enzyme to change its shape, which prevents it from doing its job, thus reducing nucleotide production in the cell.

“This is the first time that an enzyme involved in that specific pathway or function has been identified as a ppGpp target,” Wang says. “If you limit the consumption of nucleotides but not their production, the nucleotide pool is going to explode, which isn’t good for the cell. So we’ve shown that ppGpp actually addresses this problem as well.”

In addition to PurF and other enzymes required for nucleotide production, the researchers noticed that ppGpp also binds to many GTPase enzymes involved in translation. This could indicate a failsafe mechanism slowing down translation by striking multiple, similar enzymes in an almost redundant manner in the face of starvation.

As Wang continues to refine his method, he aims to increase its specificity and ensure his capture compounds bind to the exact same proteins they would inside a live cell. He also hopes to screen for ppGpp binding proteins in other bacteria, including pathogens that rely on ppGpp to survive within their hosts and propagate conditions like tuberculosis.

“This is an exciting chemical approach to better understand the function of a long-studied conserved signaling molecule in bacteria,” says Jue Wang, professor of bacteriology at the University of Wisconsin at Madison, who was not involved with the study. “Their findings and techniques are highly relevant to many other bacteria, and will greatly improve knowledge of how bacteria use this critical signaling molecule to mediate everything from surviving in the human gut to causing disease.”

Adds Laub: “We are still discovering new nucleotide-based signaling molecules in bacteria even today, and every single one of them could eventually be derivatized in a similar way to identify their binding partners.”

This research was supported by a fellowship from the Jane Coffin Childs Memorial Fund for Medical Research and a grant from the National Institutes of Health.

A Summer of Protein Degradation and the Beauty of Basic Science

MSRP-Bio student Elizabeth Bond worked in the Baker Lab, investigating the macromolecular machines that roam the cell and gobble up unneeded proteins.

Raleigh McElvery
September 25, 2018

Elizabeth Bond’s greatest summer accomplishment is proudly displayed as the background image on her phone. To the casual observer, it looks like columns of black blobs, but to Bond this stained protein gel signifies that, after two long weeks, she successfully isolated her protein of interest. The snapshot also underscores that she’s found her “people” — the kind who, as she describes, “will freak out with you over a great looking gel.”

A rising senior at UMass Amherst, Bond joined 18 fellow MIT Summer Research Program in Biology (MSRP-Bio) students — collaborating in labs across the biology department and various MIT-affiliated institutes for 10 weeks. Together, they attended seminars, lectures, Q&A sessions, meals, and field trips while living in dorms and bonding over science and life in general.

“You’re with a group of other college students looking towards the future, and you’re all stressing out about what comes next,” she says. “That’s amazing because you’re able to talk about your different insecurities and anxieties. You have a built-in support system that you might not get by staying at your home institution over the summer.”

Bond grew up not too far from MIT in the quiet town of Boxford, Massachusetts. Before setting foot on campus, she expected MIT to be cutthroat and competitive. Instead, she found “a bunch of nerds who are willing to help other nerds learn, make mistakes, and be human beings.” The researchers she met were supportive and eager to share their insights, scientific or otherwise. “In addition to being really interesting, these conversations helped me feel that I fit in with a group of very intelligent scientists,” she says.

As a biochemistry major, Bond appreciates basic science because it allows her to probe biological phenomena with no immediate goal other than to understand the underlying mechanisms. “Maybe 10 years down the line my research will help someone’s translational work, but right now I can pursue knowledge for its own sake,” she says. “The beauty of basic science is that you’re able to study things because they’re cool, while also contributing to the body of work your lab family began before you.”

At UMass Amherst, she serves as an undergraduate research assistant, investigating AAA+ proteases — the same protein degradation machines she studied all summer in Tania Baker’s lab, mentored by graduate student Kristin Zuromski. Back home, Bond examines these proteases in bacteria, using a combination of microbiology and computational biology. As a member of the Baker lab, she studied these macromolecular machines leveraging biochemical approaches.

She likens AAA+ proteases to Pac-Men from the classic arcade game, roaming the cell and gobbling up misfolded, excess, or unneeded proteins. One of the AAA+ proteases studied in the Baker lab is ClpAP, which is comprised of the AAA+ unfoldase ClpA and its partner peptidase ClpP. Bond’s protein of interest this summer was ClpA, a hexameric protein depicted as a cylinder with a central channel. ClpA unfolds and threads protein substrates through its channel, which contains pore loop structures that protrude from the chamber and play important roles in the function of ClpA. From there, the proteins enter ClpP, where they are degraded into small peptide fragments.

There are two types of pore loops in ClpA, D1 and D2, but their respective roles in the recognition, unfolding, and movement of proteins for degradation are not fully characterized. Bond hoped to discern their roles relative to one another.

She introduced a mutation into the gene that encodes ClpA, switching one amino acid for another in the D2 pore loop, in a region thought to be critical for recognizing proteins targeted for degradation. This mutation would, in theory, lead to a variant of ClpA where the D1 pore loop retained normal activity, but the D2 pore loop was unable to function. She used chemical crosslinking to generate a ClpA dimer variant that was half wildtype and half mutant in the D2 pore loops, and monitored the ability of the assembled hexameric AAA+ protease to function.

By observing the degradation activity of this crosslinked ClpA variant containing three active and three inactive D2 pore loops in an alternating order, she hoped to get a better sense of the role the D2 pore loops play in ClpAP protease function.

Although there is still more to be done to answer this particular question, reflecting on the summer Bond feels her project went “surprisingly well,” despite being more challenging than she initially anticipated — primarily due to multi-week protein purification experiments and performing many procedures simultaneously. She arrived with the sole intention of bolstering her biochemistry knowledge, and left with a greater appreciation for the breadth of scientific fields she could pursue.

“MSRP-Bio gave me the chance to talk with students and faculty members working in multiple branches of science,” she says. “I study bacteria, but I can learn a lot from someone researching roundworms or cancer cells, or using computational approaches to biology. Those conversations prompted me to think more critically about my own research.”

Besides feeling integrated into the tightly-knit Baker lab, her favorite aspect of the summer was the bond she formed within her MSRP-Bio cohort. In addition to freaking out over protein gels, they started their own journal club, and they discussed personal struggles, family, where they came from, and where they want to go.

“A lot of students of color who come from underrepresented groups in science, like I do, have this anxiety about not being smart enough or not fitting in,” she says. “The program allows you to bond over these shared feelings and that is part of what makes it really amazing for students who are trying to do great things, but do not often feel fully represented.”

At the beginning of the summer, Bond hadn’t fully admitted to herself that she wanted to apply to grad school. “It was easier for me to be ambiguous about what I wanted to do, because it was scary to admit that grad school was something I might actually want,” she recalls. After a summer at MIT, she’s gained the confidence to apply and state her ambitions out loud.

“My project’s been amazing and great, but now I want to have my own body of work,” she says. “It’s something I have this great urge to do — and, because of MSRP-Bio, I’m ready for it.”

Photo credit: Raleigh McElvery
Parasite’s riff on essential enzyme highlights unique biology
Nicole Giese Rura | Whitehead Institute
September 18, 2018

Cambridge, Mass. — The primary currency of energy in cells—adenosine triphosphate (ATP)—is essential for their survival and without it, cellular processes would seize. In the apicomplexan Toxoplasma gondii (T. gondii), a parasite that Whitehead Member Sebastian Lourido studies, key components of the ATP synthase—the enzyme responsible for ATP production—have remained elusive. While investigating indispensable proteins with unknown functions, Lourido and Diego Huet, a postdoctoral researcher in Lourido’s lab, identified a critical component of the enzyme. While highly conserved from yeast to humans, it proved to be considerably different in T. gondii. The findings, published online September 11 in the journal eLife, underscore the unique biology of these parasites and highlight differences between them and their human hosts.

More closely related to plants than to animals, the single-celled apicomplexans are among the most common and deadly human pathogens. According to the World Health Organization, every year these diseases sicken hundreds of millions, kill hundreds of thousands—primarily children—and cost billions of dollars to treat. Species of apicomplexans cause malaria (Plasmodium spp.), cryptosporidiosis (Cryptosporidium spp.), and toxoplasmosis (T. gondii).

Using a CRISPR-based genetic screen that they had adapted to T. gondii, Lourido and Huet had previously identified about 200 genes in T. gondii that are fitness-conferring and specific to apicomplexans. Of that cadre, a few were localized to the mitochondria, where cells manufacture ATP, the cellular currency of energy. Because those genes have not been annotated previously, and the proteins encoded by them have no known function, Huet ran their protein sequences through a database that compared them to protein sequences with known structures.

One of the proteins came back with an interesting hit: it shares structural similarity, but not sequence similarity, with an integral part of the ATP synthase. Most of the protein subunits that compose the apicomplexan ATP synthase have been identified, but key components of the stator—a portion of the enzyme essential for its function—was not yet known.

When Huet experimentally removed the function of the stator subunit in T. gondii, the parasites’ growth stalled, their mitochondria were misshapen and shrunken, and energy production halted—all traits typical of interrupted ATP synthase function.

Because the apicomplexan ATP synthase varies so much from its hosts’ version, those differences, like the unusual stator, could serve as future drug targets. But for Lourido, who is also an assistant professor of biology at Massachusetts Institute of Technology (MIT), the unique stator protein emphasizes how unique and extraordinary apicomplexan organisms are compared to us and their other hosts.

This work was supported by the National Institutes of Health (NIH grants 1DP5OD017892, R21AI123746, and K99AI137218).

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Sebastian Lourido’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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Full Citation:

“Identification of cryptic subunits from an apicomplexan ATP synthase”

eLife, online September 11, 2018.  DOI: 10.7554/eLife.38097

Diego Huet (1) , Esther Rajendran (2) , Giel G van Dooren (2) , Sebastian Lourido (1,3*).

1. Whitehead Institute for Biomedical Research, Cambridge, United States

2. Research School of Biology, Australian National University, Canberra, Australia

3. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States