Computer-designed ricin toxin inhibitors

November 2, 2018 § Leave a comment

To date, there is no known antidote for ricin poisoning.

Ricin is a cytotoxic protein extracted from mashed castor beans during castor oil production. It was tested in the 1940s as a chemical warfare agent by the United States military and is believed to have been used in the 1980s by some terrorist organizations. In 2014, a student at Georgetown University was arrested for manufacturing the poison in his dorm room.


Ricin is extracted from the beans of the castor oil plant.

Gerd Rocha, an associate professor at the Federal University of Paraiba in Brazil, is attempting to identify novel ricin antidotes. Rocha and colleagues are employing computational chemistry techniques to measure small-molecule binding affinities for ricin toxin. Their findings are published in the Journal of Chemical Information and Modeling.

Ricin consists of two subunits, ricin toxin A (RTA) and ricin toxin B (RTB), connected by a disulfide bond. RTB causes the protein to be absorbed into the endoplasmic reticulum of a cell, where an isomerase cleaves the disulfide bond, releasing RTA, the subunit responsible for toxicity. Once inside, RTA binds ribosomal RNA and depurinates adenine from the ribosome’s GAGA motif at the sarcin-ricin loop. Ultimately, this prevents protein synthesis and causes cell death.

RTA’s active site is a challenging target for small molecule drugs because of its large cavity and polar characteristics, which cause it to form favorable hydrogen bonding networks with water. Drug binding disrupts these networks, resulting in unfavorable energetic penalties.

“Ricin intoxication involves extra- and intracellular events, so competitive inhibitors for the RTA active site is only one of the strategies for inhibiting this potent toxin,” Rocha said. “However, with the increase of the screening libraries, such as ZINC15, which presents approximately 1 billion compounds, searching for new scaffolds for the RTA active site can still be a promising alternative, which needs to be exhaustively evaluated.”

Computational chemists use databases, like ZINC15, to explore structure-activity relationships between chemical scaffolds and protein targets through virtual screening. Virtual screening is like traditional high-throughput screening except that a computer model predicts activity instead of a binding assay. This approach allows scientists to rapidly identify interesting scaffolds that are then prioritized for experimental testing. Virtual screening’s goal is to increase efficiency of high-throughput screening for finding active compounds.

But before they can virtually screen databases like ZINC15 for new ricin antidote scaffolds, chemists have to build models capable of distinguishing ricin binders from non-binders. Rocha and colleagues designed a two-tiered model combining molecular docking and steered molecular dynamics to rank a small set of RTA inhibitors.

Molecular docking measures the interactions between a flexible ligand and rigid protein. These interactions include hydrogen bonding, hydrophobic contacts, pi-pi stacking between aromatic groups, and penalization terms (for solvent exposure or conformational strain) and are reported as a “docking score.” This score represents a molecule’s predicted binding affinity.  This study used the free docking program, AutoDock Vina.

“We attempted, as a method validation, to use the most potent (ricin) inhibitors previously described and available in the Protein Data Bank,” Rocha said. “Unfortunately, the low availability of structures of RTA-ligand complexes in the PDB is a limiting feature of our study.”

Rocha and colleagues identified six pterin scaffold ricin inhibitors. Three inhibitors were considered active with binding affinities below 70 µM; the remaining inhibitors were inactive.

“In our results, we verified that Vina score function can efficiently predict the bioactive poses of the ligands in the RTA active site,” Rocha said. However, he said, the Vina score function alone did not correctly rank ligands by their ricin binding affinity. This can generate false-positive results in a large-scale virtual screening study.

False-positives are detrimental to any virtual screening analysis and occur when an inactive compound is predicted active. Computational chemists can minimize false-positives by modifying scoring functions, a task easier said than done, or including a molecular docking post-processing technique.

Where molecular docking is considered a “fast” approach, post-processing approaches are not. These techniques employ more sophisticated functions to measure ligand binding affinities and often include “flexible” techniques that incorporate protein movement. This study used steered molecular dynamics (SMD) for post-processing.

Molecular dynamics is a time-dependent calculation enabling researchers to visualize protein and ligand interactions. However, the time scale for these calculations is too small to replicate all biological activities, like ligand binding or unbinding. Steered molecular dynamics dictates simulations toward desired biological events by applying an external force. This external force is like two tug-of-war teams. The protein is attempting to “hold” the ligand in place, while the computer model tries to pull the pterin inhibitor from the RTA binding pocket. The strength, or force, needed to separate the ligand and protein is the ligand binding affinity.

Using SMD, the researchers ranked the six ligands in terms of binding affinity and showcased an important post-docking processing tool to prioritize ligands for experimental testing. However, the model does not guarantee a ligand will have druggable characteristics in organisms, and SMD should not be used as a stand-alone screening approach for large libraries of compounds. Instead, Rocha suggests adapting a two-tiered approach that combines molecular docking and SMD.

Rocha plans to expand the model for a large-scale virtual screening study and experimentally validating the top compounds. And maybe his model will help expand the field of known small-molecule ricin binders, ultimately giving way to an inhibitor.

George Van Den Driessche ( is a graduate research assistant in the Fourches lab at North Carolina State University.

Johns Hopkins to honor Henrietta Lacks with research building

October 9, 2018 § Leave a comment

Johns Hopkins University has announced plans to name a research building on its East Baltimore campus in honor of Henrietta Lacks whose “immortal cells” have been crucial to biomedical progress over six decades, including the development of anti-tumor and anti-viral treatments and the polio vaccine.

The naming was announced by Johns Hopkins University President Ronald J. Daniels, who was joined by descendants of Lacks, during the ninth annual Henrietta Lacks Memorial Lecture on Oct. 6. Lacks and the biomedical legacy of her cells, which were taken without her consent in 1951 shortly before her death from cervical cancer, were made famous by the bestselling 2010 book “The Immortal Life of Henrietta Lacks” by Rebecca Skloot.

The world owes much to Henrietta Lacks, whose cells were removed during a biopsy in 1951 and used for research without her knowledge or approval.

“This building will be a place that stands as an enduring and powerful testament to a woman who not only was the beloved mother, grandmother and great-grandmother to generations of the Lacks family, but the genesis of generations of miraculous discoveries that have changed the landscape of modern medicine and that have benefited, in truth, the much larger family of humanity,” Daniels said.

The announcement comes just over five years after Johns Hopkins worked with members of the Lacks family and the National Institutes of Health to reach an agreement regarding approval for researchers to access the full genomic sequence of HeLa cells, which includes traits of the family’s genome.

Groundbreaking for the building, which will adjoin the university’s Berman Institute of Bioethics and will house programs that enhance participation of members of the community in biomedical research, is scheduled for 2020 and university officials expect construction to be completed in 2022.

“We say very directly to the Lacks family, thank you,” Daniels said. “Thank you for the generosity of spirit, of hopefulness, of honesty, of collaboration that has marked our partnership. Thank you for lending Henrietta Lacks’ name to our campus. And thank you for the things that we will do together to honor and celebrate her legacy.”

This post was written by John Arnst, ASBMB Today’s science writer

Van Andel Institute Trains Next Generation of Cryo-EM Structural Biologists

September 20, 2018 § 2 Comments

By George Van Den Driessche

Cryo-electron microscopy, or cryo-EM, is structural biology’s golden ticket these days. Since the game-changing technology won the 2017 chemistry Nobel Prize, it feels like there is no problem that cannot be solved by cryo-EM.

Do you have a large protein that won’t crystallize? Are you studying transporter channels or membrane signaling proteins and have no idea what the protein looks like?  Or are you designing new antiviral agents and need the viral capsid structure? Then, at least the current trend suggests, you should consider using cryo-EM, a technique that flash freezes proteins in liquid nitrogen and analyzes electron diffraction patterns to determine the molecular structure of proteins.

The Van Andel Institute, a nonprofit biomedical research facility in Grand Rapids, Michigan, invested more than $10 million dollars to install the David Van Andel Advanced Cryo-EM Suite in 2016. Last year, VAI celebrated the opening of its new facility with a conference about cryo-EM’s advancement of structural biology. This year, VAI hosted a special cryo-EM training workshop for graduate students, postdocs and new faculty. (Full disclosure: VAI awarded travel scholarships to workshop attendees, and I was one of them.)

Attendees at the Van Andel Research Institute’s cryo-EM training workshop this summer.
Van Andel Research Institute

Huilin Li, the VAI cryo-EM core director, designed the workshop so attendees could learn about cryo-EM theory and research advances on the first day and then spend the remaining time working with the microscopes, discussing protein-preparation techniques and reviewing image processing software. “This is a privately endowed research institute. We do research, collaboration, outreach and training. This workshop is part of the training,” Li said.

VAI assistant professor Juan Du, one of the presenters, studies TRPM4 channel proteins. TRPM4 is a calcium-activated nonselective (CAN) cation channel that monitors cellular charge by measuring calcium ion concentration. When the cell’s charge becomes too positive, calcium ions bind to TRPM4, and single-charged cations, such as sodium or potassium ions, flow through the channel. This process decreases the cell’s overall positive charge.

Du’s team determined the structure of TRPM4 and identified key calcium and modulator binding sites using single-particle cryo-EM. Their work appears in Nature. She advised attendees, “We are structural biologists, so we use all kinds of tools to solve problems, and very soon cryo-EM will be equal to X-ray crystallography, and, when that day comes, success will depend on who has a sample in hand. So, remember that biochemistry is the key to success.”

Keynote speaker Steve Ludtke, a professor of biochemistry and molecular biology at Baylor College of Medicine, presented his lab’s research streamlining cryo-electron tomography (cryo-ET) image annotation.

Cryo-ET allows researchers to see the dynamic interactions of frozen macromolecules and offers insight into the native environments of cells. Traditional cryo-EM requires protein purification, and this raises the possibility of disrupting a protein from its natural orientation.

Preparing grids for Titan Krios microscope: (left) loading grids prepared in liquid nitrogen, (top right) an unprepared grid, and (bottom right) the microscope
George Van Den Driessche

However, a major hurdle facing cryo-ET is the time required for image annotation. Image annotation is a task where scientists identify all the individual macromolecules by hand in a series images. A fully annotated cryo-ET image takes about a week to complete.

Ludkte is training deep learning models to overcome the image annotation time barrier. These models are capable of learning a wide-range of macromolecule features (such as distinguishing double-membrane mitochondria from single-membrane organelles) and require minimal training. Each model needs only 10 human annotated training images before the algorithm can begin recognizing patterns in the cryo-ET image.

Georgia Institute of Technology graduate student Kasahun Neselu said he enjoyed the hands-on training and was leaving feeling excited and filled with ideas to test on his own research projects.

Fatemeh Abbasi Yeganeh, a graduate student at Florida State University, said she enjoyed learning more about cryo-EM, meeting new people who share her passion for structural biology, and gaining confidence in the field.

“Currently, EM is the main focus of my Ph.D. project, and I take any opportunity to learn more and meet new people in the field,” Yeganeh said. “So, what can be better for a graduate student than attending a workshop that pays all your expenses, teaches you about your favorite field of study, and, also, gives you the opportunity to meet and talk to experts in the field?”

Top and side view of the TRPM4 calcium channel
George Van Den Driessche

George Van Den Driessche ( is a graduate research assistant in the Fourches lab at North Carolina State University.

Shadow gene gives otters, manatees susceptibility to Court-banned bug-killer’s toxicity

August 13, 2018 § Leave a comment

Sea otters are among the marine mammals that Nathan Clark and colleagues found to have lost production of an anti-oxidant enzyme that also breaks down organophosphate pesticides.
Flickr/Neil Hooting

If you’ve ever dreamed of being born anew as a sea otter or other marine mammal, you may be in for a neurotoxic surprise.

After the forerunners of modern whales, dolphins and manatees independently turned their backs on terrestrial lives tens of millions of years ago, their descendants soon lost the function of PON1, a gene whose encoded enzyme reduces oxidative damage to lipid particles in mammals’ bloodstreams. In losing the function of that gene, which still lingers, wraith-like, in their genetic code, a vast number of marine mammals also lost a second serendipitous function of that enzyme that allowed it to break down the neurotoxic metabolite of the pesticide chlorpyrifos.

More than 50 years after Dow Chemical Company introduced chlorpyrifos to the market, it continues to be one of the most widely used insecticides in the United States, although it was banned from residential use in 2000. That ban came on the heels of highly-publicized cases of chlorpyrifos poisoning in the ‘90s that left a West Virginia child paralyzed, and a Texas man in a vegetative state.

The 9th Circuit Court of Appeals in Seattle this month ordered the Environmental Protection Agency to a ban the pesticide from agricultural use within 60 days. The court’s decision, a rebuke to the agency after former EPA chief Scott Pruitt denied an environmental group’s petition to ban the use of chlorpyrifos on food crops, was made public less than an hour after a study on the marine mammals’ loss of PON1 function was coincidentally published in the journal Science.

Clement Furlong, a biochemist at the University of Washington and co-author on the paper, applauded the court’s action.

“The decision to discontinue the use of chlorpyrifos was based on very solid scientific evidence from many different laboratories, and the (initial) decision to discontinue the ban was certainly not,” Furlong said. “It’s to the benefit of sensitive humans, particularly the very young, and animals that have no protection, including the marine mammals, birds, fish, other animals that are missing the paraoxonase function.”

Experts estimate that manatees’ mammalian ancestors began adapting to aquatic lives around 60 million years ago, whereas the forebears that became both dolphins and whales returned to the ocean nearly 50 million years ago.
Flickr/Robert Engberg

In addition to sirenians, the taxonomic order containing manatees and the extinct Steller’s sea cow, and cetaceans, the order containing dolphins, whales and porpoises, the researchers found that North American beavers, as well as pinnipeds including the Weddell seal, Hawaiian monk seal and elephant seal, lost function of the PON1 gene. Senior author Nathan Clark believes this may have been an adaptation to the oxidative stresses caused by prolonged diving.

“If we pump up our bodies with tons of oxygen, submerge, deplete all that oxygen and then come back and rapidly reperfuse our bodies with oxygen, proteins and DNA and lipids would just be oxidized like crazy, and that would cause a lot of damage, and we would not survive very long,” Clark said.

To deal with the stress, marine mammals have evolved to pump out high amounts of the antioxidants catalase and superoxide dismutase.

“So one thought is that if their upfront defenses for free radical–producing things that would cause oxidative damages are so strong, then maybe PON1 is no longer necessary,” Clark said.

While it is unclear when PON1 evolved, the gene is believed to be ubiquitous among land mammals due to the role it plays in preventing the buildup of arterial plaques. Fish and birds lack PON1, and their populations have subsequently been devastated by chlorpyrifos, whose metabolite chlorpyrifos oxon disrupts acetylcholine activity at nerve terminals.

Clark and his colleagues discovered that marine mammals had lost PON1’s functionality by scouring the protein-coding sequences of more than 17,000 genes in 60 species for signs that genes had become nonfunctional pseudogenes through the addition of early stop codons and frameshift mutations. They tabulated the results in a matrix and used phylogenetic software to score each gene on how quickly it was lost in a species.

“Physiologists and marine biologists have known for decades that dolphins and whales have no sense of smell, so we thought we’d see a lot of chemosensation genes like olfactory receptors, and we did,” said Clark, a comparative genomicist at the University of Pittsburgh. “Some of our expectations didn’t come out, but then this gene Paraoxonase 1 was sitting right on top of the list.”

Margaret Hunter, a research geneticist at the U.S. Geological Survey’s Wetland and Aquatic Research Center in Gainesville, Florida, who was not an author on the study, is also intrigued at the scope of PON1’s loss of function.

“I thought it was quite surprising, and a pretty interesting find, especially because of the breadth of the mutation in the different species,” she said. “And the evolutionary divergence of all of these animals is extremely broad too, all the way to sirenians, which originated in Africa and are related to elephants and hyraxes.”

Hunter, who specializes in manatee population genetics, said the gene’s loss of function might be less heavily tied to diving, given PON1’s loss in manatees and their preference for coastal waters.

“We’ll see them sometimes at 20 feet, but they really prefer shallower waters,” she said. “They do hold their breath, so that could be related to it, but it is pretty interesting because they’re not diving or holding their breath for a length that we see in some other cetaceans or pinnipeds.”

Manatees retreat to inland, spring-fed waters, such as those at Homosassa Springs Wildlife State Park in Florida, during colder winter months
Flickr/John Flannery

Clark and his colleagues plan to expand the scope of marine mammals in their next analysis and begin collaborations with ecologists in Florida to monitor manatees for the presence of chlorpyrifos, which, despite the court-ordered ban, will likely remain in agricultural runoff in the U.S. for the near future.

“Just because you ban a pesticide doesn’t mean it’s removed from the environment very rapidly,” Hunter said. “We still need to monitor populations for these pesticides moving forward.”

This post was written by John Arnst, ASBMB Today’s science writer

Pertussislike toxins in E. coli

September 11, 2017 § 1 Comment

Cladogram of AB5 toxin sequences identified in genome sequenced E. coli strains available on the NCBI database.

Pertussislike toxins are pathologic proteins released by various bacteria during infection.  (Pertussis toxin itself has a well-established role in whooping cough.) The unique structures shared by pertussislike toxins enable them to recognize cells by binding to long carbohydrate chains, known as glycans, on the cell surface and then enter the cytoplasm.

Pertussislike toxins are less studied than pertussis itself, and their cellular mechanisms remain relatively unknown, but a recent report in the Journal of Biological Chemistry elucidates the structures and functions of a subset of them.

“Bacteria that are able to cause disease are often only able to make us sick because they produce specialized proteins that misdirect or shut down parts of our immune system,” explained Dene R. Littler at Monash University, the lead author on the JBC report. Pertussis and pertussislike toxins are among these specialized proteins. They modify G proteins, critical signaling molecules that, as Littler put it, “transduce messages to immune cells, allowing them to respond to signs of infection.”

In the JBC report, Littler and colleagues describe using computational methods to zero in on pertussislike toxins in the genomes of various pathogenic E. coli strains.  They refer to the genes as E. coli pertussislike toxins, or EcPlt for short.

They next set out to determine the function of these genes. “While many of these toxins had been described as pertussislike, previously nobody really looked to see if they function in the same way” as pertussis itself, Littler said.  The researchers, he added, were “interested in determining what had been conserved amongst these related toxins and what had changed.”

They used cell-proliferation assays to determine the cytotoxicity of EcPlt. The researchers observed that EcPlt treatment of human embryonic kidney (HEK293T) or African green monkey kidney epithelial cells (Vero) halted cell proliferation.  EcPlt also bound to glycans on the cell surface.

They determined the protein targets of EcPlt by using an ADP-ribosylation assay. ADP-ribose is a large, bulky chemical group used by many bacterial toxins to modify and disrupt host signaling proteins, including G proteins. “This creates a large protrusion on the interacting interface and prevents receptor coupling,” Littler said. The researchers found that EcPlt targets the G proteins known as Gαi/o for ADP-ribosylation to interfere with their proper signaling. Interestingly, this modification occurs at lysine and asparagine residues in Gαi/o, as opposed to the typical cysteine residue targeted by other pertussislike toxins.

Finally, the researchers investigated the structure of EcPlt in fine detail using X-ray crystallography. They report that the toxin resembles a “blunted pyramid” in which a single A subunit sits atop five copies of a B subunit. This structure is typical of so-called AB5 toxins expressed by several bacterial pathogens. These toxins are oxidized and inactive outside the cell, but they become reduced and active upon entering the cytoplasm. According to Littler, “this form of the toxin is more dynamic and is optimized for rapid enzymatic modification of human proteins.”

Littler said he hopes the team’s “active toxin structures help identify residues that are essential for activity of pertussislike toxins and help define ways to produce inactive recombinant versions.” This would facilitate vaccine production.

Additionally, he said knowing the EcPlt structure “will help with the development of small-molecule inhibitors that may aid people whose immune systems struggle to curtail infection.”

This post was written by Stefan Lukianov, a Ph.D. candidate at Harvard Medical School and contributor to ASBMB Today.



The cyanides of Titan

August 4, 2017 § Leave a comment

An artist’s rendering of the Huygens probe come to rest on one of Titan’s methane lakes.
NASA/Gregor Kervina

Vinyl cyanide isn’t a ’70s punk band, even though it sure sounds like it could have been. It’s a nitrogenous compound just confirmed by NASA researchers in the journal Science Advances to exist in the atmosphere of Saturn’s moon Titan. Some scientists in the past have proposed that vinyl cyanide might have the potential to form membranelike spheres called azotosomes.

The planetary scientists and astronomers at NASA’s Goddard Space Flight Center used calibration data from the Atacama Large Millimeter Array of telescopes in northern Chile obtained in 2014 to confirm the presence of vinyl cyanide in Titan’s atmosphere. They also estimated that quantities of the compound copious enough to reach saturation and form a solid precipitate likely exist in Ligeia Mare, Titan’s second-largest sea. Located in the north polar region of Saturn’s largest moon and named after one of the mythological Greek Sirens, the sea is about one and a half times the size of Lake Superior and made up almost entirely of methane.

Titan is roughly 50 percent larger than our moon, and its frigidity is otherworldly. The average surface temperature is more than 160 degrees lower than the coldest temperature ever recorded on Earth, which is minus 128 Fahrenheit.

At such icy temperatures, cell membranes made of phospholipid bilayers, like those surrounding cells found on Earth, would be too rigid to function properly. A more significant barrier to life on Titan is that all known cellular processes, such as the storing of genetic information as DNA, must occur in water, meaning any biochemistry on Titan would operate in a manner entirely foreign to our own.

While the presence of vinyl cyanide in Titan’s atmosphere was predicted in 2007 in a paper in the journal Icarus based on spectral data collected by the mass spectrometer on the Cassini spacecraft, the instrument wasn’t sensitive enough to definitively distinguish between vinyl cyanide and extremely similar compounds.

“With Cassini, they found evidence for a protonated form of the molecule in the mass spectrometer,” said Maureen Palmer at NASA Goddard, the first author on the new paper. However, she said, “you could have multiple different molecules with the same mass, so it can be hard to distinguish that way.”

Titan is the only moon in our solar system known to have a dense atmosphere and is the only celestial body other than Earth to have a dense atmosphere rich in nitrogen, which coats the planet in an orange-brown haze and falls to the surface with methane rain. In 2015, a group of researchers at Cornell University used supercomputer-generated simulations to propose that vinyl cyanide was capable of coalescing to form spherical membranes held together by the polarity of the nitrogen-containing groups, which are known as azoto groups. Thus, the structures were dubbed azotosomes.

“The key to vinyl cyanide is that it was able to form a stable, spherical structure in the liquid methane,” said Jonathan Lunine, a planetary scientist who co-authored the 2015 paper, also in Science Advances. “But, at the same time, it was flexible. Molecules that didn’t work were either ones that produced unstable spheres that would fall apart or where the spheres were completely rigid, which is not the way cellular membranes work.” If a cell membrane is too rigid, molecules are unable to diffuse in and out, making cellular processes essentially impossible.

“I was very pleasantly surprised that (the Goddard group) detected (vinyl cyanide),” said Lunine. “When we made our list of molecules that we examined in our calculations, our criterion was that they either had been detected in Titan’s atmosphere or that there was a tentative suggestion or tentative detection that they were there. That was true of vinyl cyanide. The Cassini ion-mass spectrometer had some indication in its spectra that it might be there, but it wasn’t definitive, so this was great. It’s very nice to see it’s really there.”

The presence of vinyl cyanide, though, doesn’t mean that the hypothetical azotosomes are coalescing and propagating life through strange exobiology in the seas of Titan.

“I kind of doubt that Earth-like genes and catalysts would be available,” said David Deamer at the University of California Santa Cruz’s Department of Biomolecular Engineering. “This is very conjectural, in my opinion. I like conjectures, by the way . . .  every conjecture can be turned into a hypothesis if you can find a way to test it.” Deamer’s research involves the origin and evolution of membrane structures.

“We’re just really depending on acts of imagination, saying, ‘Well, maybe something’s there. Let’s go look for it.’ And that’s the way science works: It’s the exploration. And these are papers exploring ideas and offering conjectural hypotheses.”

About cyanide

  • A cyanide group consists of a carbon atom covalently triple-bonded to a nitrogen atom.
  • When cyanide groups are attached to an organic carbon structure, they can become extremely useful for industrial applications such as the manufacturing of glues, rubbers and plastics. This is true of the double-bonded carbon atoms that make up the vinyl group in vinyl cyanide.
  • The infamous poisons sodium cyanide and potassium cyanide work by reacting with stomach acids to form hydrogen cyanide, which shuts down cellular respiration and ultimately cuts off oxygen to the brain.

This post was written by John Arnst, ASBMB Today’s science writer

Not your average PTPase: discovering PTEN’s substrate

July 17, 2017 § Leave a comment


In 1997, a couple of research groups discovered a gene now known as PTEN. The amino acid sequence of the protein it encoded resembled that of a protein tyrosine phosphatase, a similarity that implied PTEN might have a tumor-suppressing function. But the way the protein operated was not clear.

Jack E. Dixon, whose lab at the University of Michigan worked on protein tyrosine phosphatases, and his postdoc Tomohiko Maehama set out to determine PTEN’s substrate, which was integral to understanding how PTEN functioned. Other labs had been trying to determine what protein PTEN acted on, but with no luck. “It occurred to us, and this was I think one of the great insights, that maybe this protein tyrosine phosphatase isn’t really a protein phosphatase at all!” Dixon says. “Maybe it works on something else. And that turned out to be exactly correct.”

Dixon and Maehama discovered that in fact PTEN regulates the phosphorylation of a lipid, phosphatidylinositol 3,4,5-triphosphate (called PIP3 for short), a cell-growth-factor stimulant. PTEN was the first protein tyrosine phosphatase found to regulate a lipid instead of a protein.

The pair showed in a test tube that PTEN removes the phosphate from the 3 position on PIP3 to convert it to the nongrowth-stimulating PIP2. The resulting 1998 paper “was a particularly important one for shaping (scientists’) understanding of a key signaling pathway in normal cells,” says Eric Fearon, director of the University of Michigan Comprehensive Cancer Center. That pathway is the AKT signaling pathway, which is frequently disrupted in cancer.

Dixon and Maehama performed thin-layer chromatography and showed that PIP3 is downregulated as a result of transfecting in PTEN. “When we developed that TLC, down at the bottom of the gel, we could see PIP3 behave like we thought it would behave,” says Dixon. “That was a spectacular moment.”

By regulating PIP3, which is stimulated by insulin, PTEN serves as an important second messenger in cell signaling. “It’s like the brakes and the accelerator in a car,” Dixon says. “The accelerator is in this case insulin, and the brakes are PTEN. If you lose your brakes, you become tumorgenic.” PTEN downregulates PIP3 in cells without activating the growth enzyme phosphoinositide 3-kinase. PTEN is commonly found to be missing in cancers, including prostate and endometrial cancers; its absence is a common indicator that the tumor will grow quickly.

“Different scientific labs report different results, and when science is working well, one scientific publication is then the foundation for a subsequent one,” says Ramon Parsons, chairman of the department of oncological sciences at the Icahn School of Medicine at Mt. Sinai, who led one of the groups that discovered PTEN. Within six months, he says, a whole range of groups were able to confirm and build upon Dixon and Maehama’s findings.

Since whole-exome sequencing became possible in the past decade, Parsons says, it has become clear that PTEN is likely the most frequently mutated tumor suppressor besides P53 in all cancers.

Dixon and Maehama’s JBC paper is “certainly viewed as one of the seminal papers in the whole study of PTEN as a tumor-suppressor gene,” Fearon says.

It is now known that PTEN plays an important role outside of cancer in processes such as brain development. In fact, PTEN mutations have been tied to a subset of autism resulting from the uncontrolled growth of nerve fibers in the brain.

Dixon and Maehama’s 1998 paper “was the first paper to definitively establish the function of PTEN as a tumor-suppressor gene in cell signaling,” a critical step in exploring therapeutic effects in cancers, Fearon says. Small molecules are being studied to target defects in the AKT pathway.

“Even though it was a very short paper, I think it was the pivotal paper in highlighting the function of PTEN,” Fearon says. “It’s beautiful work, and extremely well done, which is why I think it’s stood the test of time.”

This post was written by Alexandra Taylor (alexandraataylor[at], a master’s candidate in science and medical writing at Johns Hopkins University. She writes about “Classic” articles in the Journal of Biological Chemistry. See more of her work in JBC here.

Australian spider venom helps prevent stroke damage

March 22, 2017 § Leave a comment

The moderately venomous Australian funnel-web spider Hadronyche infensa Courtesy of Bastian Rast

The moderately venomous Australian funnel-web spider Hadronyche infensa
Courtesy of Bastian Rast

Researchers at Australia’s University of Queensland have identified a peptide from spider venom that can protect mice from brain damage if it’s given up to eight hours after an ischemic stroke. The researchers presented their work this week in the journal Proceedings of the National Academy of Sciences.

The only drug available to treat strokes is tissue plasminogen activator, or tPA, which works by breaking up the clots that cause ischemic strokes. At too high of a dose, however, tPA can induce hemorrhaging. Because of this risk, the drug is utilized for only about three percent of stroke cases worldwide.

Stroke is “the second-biggest cause of mortality in the world, and we don’t really have a drug to treat these patients,” says senior author Glenn F. King the University of Queensland’s Institute for Molecular Bioscience.

Ischemic strokes are more common than blood vessel-bursting hemorrhagic strokes and occur when an obstruction in the brain’s blood vessels prevents oxygen from reaching neurons. In the absence of oxygen, the neurons begin to break down glucose by anaerobic respiration. This creates lactic acid as a byproduct. The lactic acid causes a drop in local pH that leads to toxic acidosis and cell death.

King and colleagues had previously shown that the peptide PcTx1 found in the venom of the South American tarantula Psalmopoeus cambridgei was effective in preventing cell death in mice if given up to two hours after a stroke. According to King, a Ph.D. student in his lab who was performing genetic sequencing on the venom gland of the Hadronyche infensa spider happened to identify a molecule, Hi1a. Hi1a was strikingly similar to PcTx1. H. infensa is native to Australia and a relative of the deadly Sydney funnel-web spider, but its venom is much less lethal.

Hi1a has a structure similar to two PcTx1 molecules joined together but has a different mechanism of action that makes its binding much more difficult to reverse. When Hi1a binds to an ASIC1a channel, it prevents the channel from activating, which averts the neurotoxic death cascade from occurring.

To examine Hi1a’s ability to protect neurons from stroke damage, the researchers first synthesized the peptide in bacterial cultures. They then injected it into mice at two, four or eight hours after an ischemic stroke had been induced.

“What surprised me the most was how well it worked at eight hours,” says King. Even at four hours, he says, they were able to protect the area directly surrounding the clot that’s been believed to die “very quickly and very irreversibly. That’s never really been seen before.”

Jorge Ghiso at New York University’s Langone Medical Center, who was not involved in the study, notes the peptide’s long-acting ability to protect neurons. “It’s very promising in the sense that the molecule provides a wider therapeutic window than tissue plasminogen activator to efficiently reverse the damage produced by the ischemic stroke”, he says. The peptide “has been already tested up to eight hours after stroke onset, and it works in a very low dose, which are both encouraging findings for future preclinical studies.”

King plans to examine the peptide’s activity over longer period of times, and hopes once the peptide’s ability to treat hemorrhagic strokes has next been examined, it could move into clinical trials within the next 18 months to two years. He envisions it eventually be implemented into a medication that would be a boon to rural patients who live far from medical centers.

“They’re going to get moved into a city hospital, and during that time, the brain is just dying,” he says. A drug that could treat both ischemic and hemorrhagic strokes “gives the first responders the opportunity to give the drug without any triage, and that’s going to really save a lot of neurons.”

This post was written by John Arnst, ASBMB Today’s science writer

Intrinsically disordered proteins help tardigrades survive desiccation

March 16, 2017 § Leave a comment

H. dujardini tardigrade Courtesy of Bob Goldstein and Vicky Madden at UNC Chapel Hill -

Scanning electrom microscope image of a tardigrade
Courtesy of Bob Goldstein and Vicky Madden at UNC Chapel Hill –


The humble tardigrade, an organism whose name means “slow stepper,” has long been known to survive bursts of ultraviolet radiation, freezing temperatures, the vacuum of space and extreme droughts. But, until now, the mechanisms by which these creatures do so have remained unclear. In a paper published today in the journal Molecular Cell, researchers at the University of North Carolina, Chapel Hill, report that intrinsically disordered proteins unique to tardigrades, who are also known as “water bears,” are responsible for the organisms’ ability to survive extreme desiccation.

As tardigrades dry out, they crank up their production of intrinsically disordered proteins, which lack three-dimensional structures. As the drying progresses, these proteins vitrify around internal cellular components, forming an amorphous glasslike solid.

“It’s a lot more gentle on the cell,” says lead author Thomas Boothby. The solid prevents proteins that are sensitive to desiccation from denaturing and aggregating; otherwise, these proteins would form crystals that would shred DNA and cell components once water is added back to the system.“What we envision is happening is that membranes and proteins are basically being coated in these disordered proteins that form a glassy matrix around them.”

According to Boothby, one of the competing theories has been that tardigrades use the sugar trehalose to form the glassy matrices that protect their cells. In animals that use trehalose to survive desiccation, such as brine shrimp, the sugar makes up around twenty percent of body weight; the concentration in tardigrades has  been observed at about 2 percent. “When you couple that with genetic evidence that tardigrades don’t have the enzyme to make trehalose, it makes us think that they’re probably not producing the sugar themselves. They’re probably getting a little bit of it from their food source,” says Boothby.

When the researchers ran a differential gene analysis on tardigrades that had been subjected to gradual drying, they noticed 11 cytosolic heat-soluble protein transcripts, 19 secreted heat-soluble protein transcripts and two mitochondrial heat-soluble transcripts that were significantly enriched compared with hydrated conditions. All three of these protein families are believed to encode for intrinsically disordered proteins in tardigrades.

This is the first observation that intrinsically disordered proteins confer protection against desiccation in tardigrades, though nearly all organisms contain intrinsically disordered proteins. When the researchers expressed the genes that code for the tardigrade-specific intrinsically disordered proteins in Escherichia coli and Saccharomyces cerevisiae, they found that the organisms exhibited a hundredfold increase in their ability to tolerate desiccation.

“The finding that tardigrade disordered proteins are crucial for the ability of the members of the animal kingdom to survive during extreme desiccation concurs with previous work on the plant desiccation resistance that was shown to be critically dependent on several specific intrinsically disordered proteins,” says Vladimir Uversky at the University of South Florida. “The ability of tardigrade disordered proteins to vitrify represents a novel intrinsic-disorder-based molecular mechanism of protection of biological material from desiccation.”

Boothby and colleagues also noted that when tardigrades were subjected to freezing conditions instead of desiccation, the organisms activated an entirely different set of genes.

Boothby and colleagues are currently exploring the differences between which genes tardigrades activate for different harsh conditions. “Figuring out if they have just general tricks for surviving all these different stresses or if they use specific mechanisms to survive each individual stress is a really interesting question,” he says. “(It) can help us to understand how these different stress tolerances evolved as well as how the animals do them.”

This post was written by John Arnst, ASBMB Today’s science writer. 

Zebrafish display degrees of masculinization in hot water

January 26, 2017 § 1 Comment

Domesticated zebrafish Credit: NICHD/ J. Swan

Domesticated zebrafish
Credit: NICHD/ J. Swan

Sex is determined in mammals, birds and a subset of fish, primarily by a pair of chromosomes known as the sex chromosomes. Wild-type zebrafish have sex chromosomes but their domesticated counterparts depend on polygenic sex determination, in which the responsible genetic factors for sex are distributed across the whole genome. Polygenic sex determination makes sexual differentiation more unstable because it permits environmental cues to play a greater role in sexual development. However, polygenic sex determination is less understood than sex-chromosomal determination.

In a paper published in Proceedings of the National Academies of Sciences on Jan. 23, researchers at the Temasek Life Sciences Laboratory in Singapore and Institute of Marine Sciences in Spain, have examined the transcriptomal changes that occur when domesticated female zebrafish transition into males in response to warm water. A transcriptome consists of the total mRNA in a cell that codes for proteins.

Timothy Karr, a developmental biologist at Arizona State University who was not involved in the study describes it “as one of the first studies of its kind.”

Zebrafish are native to the Indian subcontinent and, for more than 40 years, have been used as a model organism for biological research. While many fish display sexual plasticity due to environmental cues, domesticated zebrafish in this study are the first to be observed to retain female gonads while displaying male reproductive genes and proteins, rather than transitioning fully to something known as a neomale. “A neomale is an individual that’s genetically programmed to become a female, but as a result of the temperature treatment, becomes male,” says László Orbán at the Temasek Life Sciences Laboratory.

The instability of polygenic sex determination in zebrafish came across as an unintentional side-effect of cultivating distinct familial lines for research over the past four decades.

“Somehow, the sex chromosomes have been lost during the domestication process,” says Orbán. While there had been controversy as to whether zebrafish sex was more strongly determined by inherited chromosomes or polygenic cues, the split between wild and domestic families was confirmed about two years ago. Researchers examined zebrafish they had retrieved from northern India and found that the wild fish still displayed sex-chromosomal determination.

Within domesticated zebrafish, the family lines develop different ratios of males to females. Francesc Piferrer’s lab at the Institute of Marine Sciences, which had previously examined the effects of temperature on fish sex ratios and helped design the study, subjected a variety of zebrafish families to water at 36° C during a window of 18 and 32 days post-fertilization. The Orbán lab members then used microarrays to identify the differences in transcriptomes between the zebrafish males and females that been experienced control and heat-exposed conditions.

Examining the transcriptomes of these fish allowed the researchers to then identify which had become neomales or pseudofemales which have ovaries but a male-like transcriptome. The researchers found that the pseudofemales displayed gonadal transcriptomes that only differed from genuine male transcriptomes by a few thousand genes. “It looks like a reprogramming process that doesn’t complete,” says Orbán. “The details are not known to us so there’s a whole area of science opening up here.”

“If it can be replicated, the authors’ claim to have discovered ‘male-like’ transcriptomes in females with morphologically developed ovaries, would be an extraordinary finding, but perhaps not for the reasons the authors envision in this study,” Karr notes.  “It would be one of the most persuasive arguments” against the dominance of chromosome-based sex determination in developmental and evolutionary biology.

This post was written by John Arnst, ASBMB Today’s science writer.