GN/ A new era of mitochondrial genome editing has begun
Genetics biweekly vol.27, 20th April — 4th May
TL;DR
- A new era of mitochondrial genome editing has begun. Scientists successfully achieve A to G base conversion, the final missing piece of the puzzle in gene-editing technology.
- To get to the places where they are needed, immune cells not only squeeze through tiny pores. They even overcome wall-like barriers of tightly packed cells. Scientists have now discovered that cell division is key to their success. Together with other recent studies, their findings give the full picture of a process just as important for healing as for the spread of cancer.
- Researchers have identified key drivers of T cell development which promote resilience to influenza virus infection.
- Beta-hydroxybutyrate, an alternative-energy molecule produced by the body in response to starvation or low-carb diets, strongly suppresses the growth of colorectal tumors in lab experiments, according to a new study.
- Bloodworms are known for their unusual fang-like jaws, which are made of protein, melanin, and concentrations of copper not found elsewhere in the animal kingdom. Scientists have observed how these worms use copper harvested from marine sediments to form their jaws, and the process may be even more unusual than the teeth themselves.
- Research shows how a protein complex, called chromatin assembly factor-1, controls genome organization to maintain lineage fidelity.
- Biologists have discovered an aberrant protein that’s deadly to bacteria. This erroneously built protein mimics the action of aminoglycosides, a class of antibiotics. The newly discovered protein could serve as a model to help scientists unravel details of those drugs’ lethal effects on bacteria — and potentially point the way to future antibiotics.
- Scientists have identified a protein that allows the fungus which causes white mold stem rot in more than 600 plant species to overcome plant defenses. Knowledge of this protein, called SsPINE1, could help researchers develop a new, more precise system of control measures for the Sclerotinia sclerotiorum fungus, which attacks potatoes, soybeans, sunflowers, peas, lentils, canola, and many other broad leaf crops.
- A new study examines immune system diversity in the critically endangered Wyoming toad and finds that genetic bottlenecks could impact a species’ ability to respond to new pathogens. The findings could inform captive breeding strategies for endangered animal populations.
- Researchers have discovered a gene responsible for prenatal death when critical epigenetic instructions are missing from egg cells. The study shows that in mice, failed epigenetic suppression of an X-chromosome gene called Xist leads to miscarriage and developmental abnormalities. Forced suppression of maternal Xist rescued the failed miscarriages.
- And more!
Overview
Genetic technology is defined as the term which includes a range of activities concerned with the understanding of gene expression, advantages of natural genetic variation, modifying genes and transferring genes to new hosts. Genes are found in all living organisms and are transferred from one generation to the next. Gene technology encompasses several techniques including marker-assisted breeding, RNAi and genetic modification. Only some gene technologies produce genetically modified organisms.
Modern genetic technologies like genome editing would not be possible without all the previous generations of genetic technologies that have enabled scientists to discover what genes are, what they do and how DNA can be modified to add, remove or replace genes. You can find major genetic technologies development milestones via the link.
Gene Technology Market
- The valuation of the genetic engineering market is projected to escalate to USD 6.90 MN by the end of 2027.
- Global Genetic Engineering Market is projected to grow at 12.48% CAGR during the assessment period (2017–2027).
- North America holds the largest share in the global genetic engineering market, followed by Europe and the Asia Pacific, respectively.
- Another research provider, MarketsandMarkets, forecasts the genome editing, genome engineering market to grow from USD 3.19 billion in 2017 to USD 6.28 billion by 2022, at a compounded annual growth rate (CAGR) of 14.5% during the forecast period. The key factors propelling market growth are rising government funding and growth in the number of genomics projects, high prevalence of infectious diseases (like COVID-19) and cancer, technological advancements, increasing production of genetically modified (GM) crops, and growing application areas of genomics.
Latest News & Research
Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases
by Sung-Ik Cho, Seonghyun Lee, Young Geun Mok, Kayeong Lim, Jaesuk Lee, Ji Min Lee, Eugene Chung, Jin-Soo Kim in Cell
Researchers from the Center for Genome Engineering within the Institute for Basic Science developed a new gene-editing platform called transcription activator-like effector-linked deaminases, or TALED. TALEDs are base editors capable of performing A-to-G base conversion in mitochondria. This discovery was a culmination of a decades-long journey to cure human genetic diseases, and TALED can be considered to be the final missing piece of the puzzle in gene-editing technology.
From the identification of the first restriction enzyme in 1968, the invention of polymerase chain reaction (PCR) in 1985, and the demonstration of CRISPR-mediated genome editing in 2013, each new breakthrough discovery in biotechnology further improved our ability to manipulate DNA, the blueprint of life. In particular, the recent development of the CRISPR-Cas system, or “genetic scissors,” has allowed for comprehensive genome editing of living cells. This opened new possibilities for treating previously incurable genetic diseases by editing the mutations out of our genome.
While gene editing was largely successful in the nuclear genome of the cells, however, scientists have been unsuccessful in editing the mitochondria, which also have their own genome. Mitochondria, the so-called “powerhouse of the cells,” are tiny organelles in cells that serve as energy-generating factories. As it is an important organelle for energy metabolism, if the gene is mutated, it causes serious genetic diseases related to energy metabolism.
Director KIM Jin-Soo of the Center for Genome Engineering explained, “There are some extremely nasty hereditary diseases arising due to defects in mitochondrial DNA. For example, Leber hereditary optic neuropathy (LHON), which causes sudden blindness in both eyes, is caused by a simple single point mutation in mitochondrial DNA.” Another mitochondrial gene-related disease includes mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), which slowly destroys the patient’s brain. Some studies even suggest abnormalities in mitochondrial DNA may also be responsible for degenerative diseases such as Alzheimer’s disease and muscular dystrophy.
The mitochondrial genome is inherited from the maternal line. There are 90 known disease-causing point mutations in mitochondrial DNA, which in total affects at least 1 in 5,000 individuals. Many existing genome editing tools could not be used due to limitations in the method of delivery to mitochondria. For example, the CRISPR-Cas platform is not applicable for editing these mutations in mitochondria, because the guide RNA is unable to enter the organelle itself.
“Another problem is that there is a dearth of animal models of these mitochondrial diseases. This is because it is currently not possible to engineer mitochondrial mutations necessary to create animal models,” Director Kim added. “Lack of animal models makes it very difficult to develop and test therapeutics for these diseases.”
As such, reliable technology to edit mitochondrial DNA is one of the last frontiers of genome engineering that must be explored in order to conquer all known genetic diseases, and the world’s most elite scientists have endeavored for years to make it a reality.
In 2020, researchers led by David R. LIU of the Broad Institute of Harvard and MIT created a new base editor named DddA-derived cytosine base editors (DdCBEs) that can perform C-to-T conversion from DNA in mitochondria. This was made possible by creating a new gene-editing technology called base editing, which converts a single nucleotide base into another without breaking the DNA. However, this technique also had its limitations. Not only is it restricted to C-to-T conversion, but it is mostly limited to the TC motif, making it effectively a TC-TT converter. This means that it can correct only 9 out of 90 (= 10%) confirmed pathogenic mitochondrial point mutations. For the longest time, the A-to-G conversion of mitochondrial DNA was thought to be impossible.
First author CHO Sung-Ik said, “We began to think of ways to overcome these limitations. As a result, we were able to create a novel gene-editing platform called TALED that can achieve A-to-G conversion. Our new base editor dramatically expanded the scope of mitochondrial genome editing. This can make a big contribution not only to making a disease model but also to developing a treatment.” As of note, being able to perform A-to-G conversions in human mtDNA alone could correct 39 (= 43%) out of the 90 known pathogenic mutations.
The researchers created TALED by fusing three different components. The first component is a transcription activator-like effector (TALE), which is capable of targeting a DNA sequence. The second component is TadA8e, an adenine deaminase for facilitating A-to-G conversion. The third component, DddAtox, is a cytosine deaminase that makes the DNA more accessible to TadA8e.
One interesting aspect of TALED is TadA8e’s ability to perform A-to-G editing in mitochondria, which possess double-stranded DNA (dsDNA). This is a mysterious phenomenon, as TadA8e is a protein that is known to be specific to only single-stranded DNA. Director Kim said, “No one has thought of using TadA8e to perform base editing in mitochondria before, since it is supposed to be specific to only single-stranded DNA. It was this thinking outside of the box approach that has really helped us to invent TALED.”
The researchers theorized that DddAtox allows dsDNA to be accessible by transiently unwinding the double-strand. This fleeting but temporary time window allows TadA8e, a super fast-acting enzyme, to quickly make the necessary edits. In addition to tweaking the components of TALED, the researchers also developed a technology that is capable of both A-to-G and C-to-T base editing simultaneously, as well as A-to-G base editing only.
The group demonstrated this new technology by creating a single cell-derived clone containing desired mtDNA edits. In addition, TALEDs were found to be neither cytotoxic nor cause instability in mtDNA. Also, there was no undesirable off-target editing in nuclear DNA and very few off-target effects in mtDNA. The researchers now aim to further improve the TALEDs by increasing the editing efficiency and specificity, eventually paving the way to correct disease-causing mtDNA mutations in embryos, fetuses, newborns, or adult patients. The group is also focusing on developing TALEDs suitable for A-to-G base editing in chloroplast DNA, which encodes essential genes in photosynthesis in plants.
Cell division in tissues enables macrophage infiltration
by Maria Akhmanova, Shamsi Emtenani, Daniel Krueger, Attila Gyoergy, Mariana Guarda, Mikhail Vlasov, Fedor Vlasov, Andrei Akopian, Aparna Ratheesh, Stefano De Renzis, Daria E. Siekhaus in Science
To get to the places where they are needed, immune cells not only squeeze through tiny pores. They even overcome wall-like barriers of tightly packed cells. Scientists at the Institute of Science and Technology Austria (ISTA) have now discovered that cell division is key to their success. Together with other recent studies, their findings give the full picture of a process just as important for healing as for the spread of cancer.
Imagine a stone wall in the countryside. Tightly packed, one stone sits on top of the other filling the tiniest gaps. A seemingly unbreachable obstacle. On their way throughout the body to fight infections, immune cells face such barriers in the form of cell-dense tissues. To do their job as the body’s rescue service, they need to find a way through. In a recent study, scientists from ISTA’s Siekhaus group together with collaborators from the European Molecular Biology Laboratory (EMBL) and three students from a local High School, took a close look at how this happens in fruit fly embryos.
During the development of these tiny, transparent animals, macrophages, the dominant form of immune cells in fruit flies, infiltrate tissues. Using high-end microscopes, the scientists were able to follow their journey. “The macrophages arrive at the wall and look for the right place to enter,” explains Maria Akhmanova, until recently a postdoc at Daria Siekhaus’ research group and first author of the study.
Cues that guide the macrophages have directed them to the right spot. There, the pioneer macrophage, the first cell to move in, is waiting. Suddenly, a part of the wall starts to move. The cell right in front of the macrophage rounds up, preparing to divide — a normal part of its cell cycle. “This is what the pioneer has been waiting for,” says Akhmanova. Moving its cell nucleus ahead, the pioneer cell now pushes forward while all the other macrophages follow in its tracks. As the Siekhaus group also recently discovered, to break through the pioneer gets an extra boost of energy through a complex process governed by a newly discovered protein the scientists named Atossa. Furthermore, the scientists learned that to shield their sensitive nucleus from damage, the macrophages develop protective armor made from actin filaments.
By precisely inhibiting, slowing down, and speeding up the division specifically of the flanking tissue cells, the researchers were now able to prove that the crucial component that allows immune cells to enter is in fact surrounding cell division. As it rounds up to prepare for division, the tissue cell at the entry site loses some of its connection points to its surroundings, the researchers observed through live imaging. In collaboration with the De Renzis lab at EMBL, the researchers also artificially induced rounding through a cutting edge technique using light to induce genetic changes. This wasn’t sufficient to get the macrophages to enter. But genetically reducing the amount of the cell connections was. “It was very exciting to see how the macrophages were only able to enter the tissue when the tissue cell lost its connections,” says Akhmanova.
“Cell division being the key process that controls macrophage infiltration is really a very elegant concept with powerful implications,” Professor Daria Siekhaus enthuses. The same mechanism that helps macrophages enter tissues could also be essential for many other types of immune cells in vertebrates like humans. In the long run, the scientists are eager to learn if manipulating the connections or the divisions of the tissue cells could help increase immune cells’ infiltration of tumors to fight them from within or help reduce immune cells’ ability to attack tissues during autoimmunity. “Our findings will also affect any researcher who is working on any migrating cell in the context of the body,” the cell biologist explains.
β-Hydroxybutyrate suppresses colorectal cancer
by Oxana Dmitrieva-Posocco, Andrea C. Wong, et al in Nature
A molecule produced in the liver in response to low-carb “ketogenic” diets has a powerful effect in suppressing colorectal tumor growth and may be useful as a preventive and treatment of such cancers, according to a new study from researchers at the Perelman School of Medicine at the University of Pennsylvania.
In the study, researchers initially found that mice on low-carb, high-fat ketogenic diets have a striking resistance to colorectal tumor development and growth. The scientists then traced this effect to beta-hydroxybutyrate (BHB), a small organic molecule produced in the liver in response to keto diets or starvation.
“Our findings suggest that this natural molecule, BHB, could someday become a standard part of colorectal cancer care and prevention,” said study co-senior author Maayan Levy, PhD, an assistant professor of Microbiology at Penn Medicine, whose laboratory collaborated with the lab of Christoph Thaiss, PhD, also an assistant professor of Microbiology. The study’s first author was Oxana Dmitrieva-Posocco, PhD, a postdoctoral researcher in Levy’s lab.
Colorectal cancer is one of the most common cancer types and kills more than 50,000 Americans annually, making it the country’s third leading cause of cancer mortality. Alcohol use, obesity, red meat, and low-fiber and high-sugar diets have all been linked to greater colorectal cancer risk.
In the study, Levy, Thaiss and their teams set out to determine, with experiments in mice, whether different types of diet could inhibit colorectal cancer development and growth. They put six groups of mice on diets that had varying fat-to-carb ratios, and then used a standard chemical technique that normally induces colorectal tumors. They found that the two most ketogenic diets, with 90 percent fat-to-carb ratios — one used lard (pig fat), the other Crisco (mostly soybean oil) — prevented colorectal tumor development in most of the animals on those diets. By contrast, all the animals on the other diets, including low-fat, high-carb diets, developed tumors. Even when the researchers started the mice on these diets after colorectal tumors had started growing, the diets showed a “treatment effect” by markedly slowing further tumor growth and proliferation.
In subsequent experiments, the scientists determined that this tumor suppression is associated with a slower production, by stem cells, of new epithelial cells lining the colon. Ultimately, they traced this gut-cell growth slowdown to BHB — normally produced by the liver as part of a “starvation response,” and triggered in this case by the low-carb keto diets.
BHB is known to work as an alternative fuel source for key organs in low-carb conditions. However, the researchers showed that it is not only a fuel source but also a potent growth-slowing signal, at least for gut-lining cells. They were able to reproduce the tumor-suppressing effects of the keto diets simply by giving the mice BHB, either in their water or via an infusion mimicking the liver’s natural secretion of the molecule.
The team showed that BHB exerts its gut-cell growth-slowing effect by activating a surface receptor called Hcar2. This in turn stimulates the expression of a growth-slowing gene, Hopx. Experiments with gut-lining cells from humans provided evidence that BHB has the same growth-slowing effect on these cells, via the human versions of Hcar2 and Hopx. Colorectal tumor cells that don’t express these two genes were not responsive to BHB treatment, suggesting their utility as possible predictors of treatment efficiency.
“Clinical trials of BHB supplementation are needed before any recommendation can be made about its use in prevention or treatment,” Thaiss said.
The researchers are now setting up just such a clinical trial of BHB — which is widely available as a dieting supplement — in colorectal cancer patients. They are also continuing to study BHB’s potential anticancer effects in other parts of the body, and are investigating the effects of other molecules produced under ketogenic conditions.
A multi-tasking polypeptide from bloodworm jaws: Catalyst, template, and copolymer in film formation
by William R. Wonderly, Tuan T.D. Nguyen, Katerina G. Malollari, Daniel DeMartini, Peyman Delparastan, Eric Valois, Phillip B. Messersmith, Matthew E. Helgeson, J. Herbert Waite in Matter
Bloodworms are known for their unusual fang-like jaws, which are made of protein, melanin, and concentrations of copper not found elsewhere in the animal kingdom. Scientists have observed how these worms use copper harvested from marine sediments to form their jaws, and the process, described in research, may be even more unusual than the teeth themselves.
Because the worms only form their jaws once, they need to be strong and tough enough to last the entirety of the animal’s five-year lifespan. They use them to bite prey, sometimes puncturing straight through an exoskeleton, and inject venom that paralyzes victims.
“These are very disagreeable worms in that they are ill tempered and easily provoked,” says co-author Herbert Waite, a biochemist at University of California, Santa Barbara. “When they encounter another worm, they usually fight using their copper jaws as weapons.”
Waite’s lab has been studying bloodworms for 20 years, but it was only recently that they were able to observe the chemical process that forms a jaw-like material from start to finish. The worm begins with a protein precursor, which recruits copper to concentrate itself into a viscous, protein-rich liquid that is high in copper and phase-separates from water. The protein then uses the copper to catalyze the conversion of the amino acid derivative DOPA into melanin, a polymer that, combined with protein, gives the jaw mechanical properties that resemble manufactured metals.
Through this process, the worm is able to easily synthesize a material that, if created in a lab, would be a complicated process involving many different apparatuses, solvents, and temperatures. “We never expected protein with such a simple composition, that is, mostly glycine and histidine, to perform this many functions and unrelated activities,” says Waite.
The team hopes that a better understanding of how the bloodworm conducts its self-contained processing laboratory could help to streamline parts of production that would benefit industry. “These materials could be road signs for how to make and engineer better consumer materials,” says Waite.
Regulation of chromatin accessibility by the histone chaperone CAF-1 sustains lineage fidelity
by Reuben Franklin, Yiming Guo, Shiyang He, Meijuan Chen, Fei Ji, Xinyue Zhou, David Frankhouser, Brian T. Do, Carmen Chiem, Mihyun Jang, M. Andres Blanco, Matthew G. Vander Heiden, Russell C. Rockne, Maria Ninova, David B. Sykes, Konrad Hochedlinger, Rui Lu, Ruslan I. Sadreyev, Jernej Murn, Andrew Volk, Sihem Cheloufi in Nature Communications
Understanding the molecular mechanisms that specify and maintain the identities of more than 200 cell types of the human body is arguably one of the most fundamental problems in molecular and cellular biology, with critical implications for the treatment of human diseases. Central to the cell fate decision process are stem cells residing within each tissue of the body.
When stem cells divide, they have the remarkable ability to choose to self-renew — that is, make a copy of themselves — or mature into defined lineages. How a specific lineage identity is maintained every time a stem cell divides can now be better understood thanks to the work of a team led by biochemists at the University of California, Riverside. The study led by Sihem Cheloufi and Jernej Murn, both assistant professors in the Department of Biochemistry, shows how a protein complex, called chromatin assembly factor-1, or CAF-1, controls genome organization to maintain lineage fidelity.
Each time a cell divides, it has to create a replica of its genome — not only its DNA sequence but also how the DNA is packaged with proteins into chromatin. Chromatin is organized into genomic sites that are either open and easily accessible or more densely packed and less accessible (or closed).
“Identities of different cells rely heavily on the genome sites that are more open because only genes located in those regions can potentially become expressed and turned into proteins,” Cheloufi explained.
She added that to maintain cell identity during cell division, the locations of open and closed chromatin, or “chromatin organization,” must be faithfully passed onto the new replica of the genome, a task largely entrusted to CAF-1.
“To help CAF-1 secure correct chromatin organization during cell division, a host of transcription factors are attracted to open regions in a DNA sequence-specific manner to serve as bookmarks and recruit transcription machinery to correct lineage-specific genes, ensuring their expression,” she said. “We wondered about the extent to which CAF-1 is required to maintain cell-specific chromatin organization during cell division.”
The authors took as a study paradigm immature blood cells that can either self-renew or turn into neutrophils, which are non-dividing cells that present our body’s first line of defense against pathogens. Intriguingly, they found CAF-1 to be essential not only for maintaining the self-renewal of these immature blood cells, but for preserving their lineage identity. Even a moderate reduction of CAF-1 levels caused the cells to forget their identity and adopt a mixed lineage stage.
“Neutrophil stem cells missing CAF-1 become more plastic, co-expressing genes from different lineages, including those of red blood cells and platelets,” Cheloufi said. “This is very intriguing from a developmental biology perspective.”
At the molecular level, the team found that CAF-1 normally keeps specific genomic sites compacted and inaccessible to specific transcription factors, especially one called ELF1.
“By looking at chromatin organization, we found a whole slew of genomic sites that are aberrantly open and attract ELF1 as a result of CAF-1 loss,” Murn said. “Our study further points to a key role of ELF1 in defining the fate of several blood cell lineages.”
The UCR researchers used immature blood cells derived from mouse bone marrow and engineered for growth in tissue culture. They validated their findings in vivo using a mouse model in collaboration with Andrew Volk, a hematology expert at the Cincinnati Children’s Hospital Medical Center and a co-corresponding author on the study. Next, Cheloufi and her colleagues would like to understand the mechanism by which CAF-1 preserves the chromatin state at specific sites and whether this process works differently across different cell types.
“Like a city, the genome has its landscape with specific landmarks,” Cheloufi said. “It would be interesting to know how precisely CAF-1 and other molecules sustain the genome’s ‘skyline.’ Solving this problem could also help us understand how the fate of cells could be manipulated in a predictive manner. Given the fundamental role of CAF-1 in packaging the genome during DNA replication, we expect it to act as a general gatekeeper of cellular identity. This would in principle apply to all dividing cells across numerous tissues, such as cells of the intestine, skin, bone marrow, and even the brain.”
A polypeptide model for toxic aberrant proteins induced by aminoglycoside antibiotics
by Mangala Tawde, Abdelaziz Bior, Michael Feiss, Feiyue Teng, Paul Freimuth in PLOS ONE
Biologists at the U.S. Department of Energy’s Brookhaven National Laboratory and their collaborators have discovered an aberrant protein that’s deadly to bacteria. In a paper, the scientists describe how this erroneously built protein mimics the action of aminoglycosides, a class of antibiotics. The newly discovered protein could serve as a model to help scientists unravel details of those drugs’ lethal effects on bacteria — and potentially point the way to future antibiotics.
“Identifying new targets in bacteria and alternative strategies to control bacterial growth is going to become increasingly important,” said Brookhaven biologist Paul Freimuth, who led the research. Bacteria have been developing resistance to many commonly used drugs, and many scientists and doctors have been concerned about the potential for large-scale outbreaks triggered by these antibiotic-resistant bacteria, he explained.
“What we’ve discovered is a long way from becoming a drug, but the first step is to understand the mechanism,” Freimuth said. “We’ve identified a single protein that mimics the effect of a complex mixture of aberrant proteins made when bacteria are treated with aminoglycosides. That gives us a way to study the mechanism that kills the bacterial cells. Then maybe a new family of inhibitors could be developed to do the same thing.”
The Brookhaven scientists, who normally focus on energy-related research, weren’t thinking about human health when they began this project. They were using E. coli bacteria to study genes involved in building plant cell walls. That research could help scientists learn how to convert plant matter (biomass) into biofuels more efficiently. But when they turned on expression of one particular plant gene, enabling the bacteria to make the protein, the cells stopped growing immediately.
“This protein had an acutely toxic effect on the cells. All the cells died within minutes of turning on expression of this gene,” Freimuth said.
Understanding the basis for this rapid inhibition of cell growth made an ideal research project for summer interns working in Freimuth’s lab.
“Interns could run experiments and see the effects within a single day,” he said. And maybe they could help figure out why a plant protein would cause such dramatic damage.
“That’s when it really started to get interesting,” Freimuth said.
The group discovered that the toxic factor wasn’t a plant protein at all. It was a strand of amino acids, the building blocks of proteins, that made no sense. This nonsense strand had been churned out by mistake when the bacteria’s ribosomes (the cells’ protein-making machinery) translated the letters that make up the genetic code “out of phase.” Instead of reading the code in chunks of three letters that code for a particular amino acid, the ribosome read only the second two letters of one chunk plus the first letter of the next triplet. That resulted in putting the wrong amino acids in place.
“It would be like reading a sentence starting at the middle of each word and joining it to the first half of the next word to produce a string of gibberish,” Freimuth said.
The gibberish protein reminded Freimuth of a class of antibiotics called aminoglycosides. These antibiotics force ribosomes to make similar “phasing” mistakes and other sorts of errors when building proteins. The result: all the bacteria’s ribosomes make gibberish proteins.
“If a bacterial cell has 50,000 ribosomes, each one churning out a different aberrant protein, does the toxic effect result from one specific aberrant protein or from a combination of many? This question emerged decades ago and had never been resolved,” Freimuth said.
The new research shows that just a single aberrant protein can be sufficient for the toxic effect. That wouldn’t be too farfetched. Nonsense strands of amino acids can’t fold up properly to become fully functional. Although misfolded proteins get produced in all cells by chance errors, they usually are detected and eliminated completely by “quality control” machinery in healthy cells. Breakdown of quality control systems could make aberrant proteins accumulate, causing disease.
The next step was to find out if the aberrant plant protein could activate the bacterial cells’ quality control system — or somehow block that system from working. Freimuth and his team found that the aberrant plant protein indeed activated the initial step in protein quality control, but that later stages of the process directly required for degradation of aberrant proteins were blocked. They also discovered that the difference between cell life and death was dependent on the rate at which the aberrant protein was produced.
“When cells contained many copies of the gene coding for the aberrant plant protein, the quality control machinery detected the protein but was unable to fully degrade it,” Freimuth said. “When we reduced the number of gene copies, however, the quality control machinery was able to eliminate the toxic protein and the cells survived.”
The same thing happens, he noted, in cells treated with sublethal doses of aminoglycoside antibiotics. “The quality control response was strongly activated, but the cells still were able to continue to grow,” he said.
These experiments indicated that the single aberrant plant protein killed cells by the same mechanism as the complex mixture of aberrant proteins induced by aminoglycoside antibiotics. But the precise mechanism of cell death is still a mystery.
“The good news is that now we have a single protein, with a known amino acid sequence, that we can use as a model to explore that mechanism,” Freimuth said.
Scientists know that cells treated with the antibiotics become leaky, allowing things like salts to seep in at toxic levels. One hypothesis is that the misfolded proteins might form new channels in cellular membranes, or alternatively jam open the gates of existing channels, allowing diffusion of salts and other toxic substances across the cell membrane.
“A next step would be to determine structures of our protein in complex with membrane channels, to investigate how the protein might inhibit normal channel function,” Freimuth said.
That would help advance understanding of how the aberrant proteins induced by aminoglycoside antibiotics kill bacterial cells — and could inform the design of new drugs to trigger the same or similar effects.
Transcriptome annotation reveals minimal immunogenetic diversity among Wyoming toads, Anaxyrus baxteri
by Kara B. Carlson, Dustin J. Wcisel, Hayley D. Ackerman, Jessica Romanet, Emily F. Christiansen, Jennifer N. Niemuth, Christina Williams, Matthew Breen, Michael K. Stoskopf, Alex Dornburg, Jeffrey A. Yoder in Conservation Genetics
A new study from North Carolina State University examines immune system diversity in the critically endangered Wyoming toad and finds that genetic bottlenecks could impact a species’ ability to respond to new pathogens. The findings could inform captive breeding strategies for endangered animal populations.
The Wyoming toad, Anaxyrus baxteri, suffered a severe population decline throughout the latter part of the 20th century due to factors including habitat destruction and fungal infection. The toad was brought into a captive breeding program in the 1990s in order to save the species. Scientists estimate a current wild population of only 400 to 1,500 animals, meaning that the toad is considered critically endangered.
“Population reduction in this species created a genetic bottleneck to begin with, meaning the level of genetic diversity is already very small,” says Jeff Yoder, professor of comparative immunology at NC State and co-corresponding author of a paper describing the work. “This is the first study to look specifically at genetic diversity in the immune systems of these toads and how it could impact them as a population.”
Yoder, with co-corresponding author Alex Dornburg of the University of North Carolina at Charlotte, performed RNA sequencing on immune tissues from three healthy, retired Wyoming toad breeders. Study co-author Michael Stoskopf, who was on the Wyoming Toad Recovery Implementation Team established in 2008, obtained the samples.
“We were focused specifically on sequences encoding toll-like receptors — TLRs — and the proteins of the major histocompatibility complex, or MHC, expressed in these tissues,” says Kara Carlson, first author of the study and current Ph.D. candidate at NC State. “These sets of genes are major components of the immune system.”
TLRs are the first responders of the immune system, and are similar, or well-conserved, between species. The MHC, on the other hand, is a large and diverse group of genes that varies between species and individuals. It can determine why one group is more resistant to a particular pathogen than another.
“MHC genes are some of the most rapidly evolving sequences in the genome,” Carlson says. “So in a healthy population there’s a lot of variety that gets passed along to descendants, enabling the species at large to adapt to different pathogens. However, if disease survivors do so because of their MHC, then that group would have a similar MHC.
“The Wyoming toads that were brought into captivity to save the species were all able to resist the fungus that had decimated the population, but that could mean that their immune diversity is reduced.”
The researchers compared the TLR and MHC of the three Wyoming toads to each other, as well as to samples from a common toad and a cane toad. Both the common toad and the cane toad showed more MHC diversity than the Wyoming toad, even though the cane toad underwent a similar genetic bottleneck.
“The small sample size in this study — which was unavoidable due to the endangered status of the toad — nevertheless lays an important framework for conservation,” Carlson says.
“Amphibians in general don’t have as many genomic resources as other organisms,” Yoder says. “And captive breeding from a small population further decreases genetic diversity. But while these toads may be better protected against the fungal infection that nearly wiped them out, they may not be equipped to deal with new pathogens down the road.”
“While we weren’t necessarily surprised by the lack of immunogenic diversity in the Wyoming toad, it does spark an important question,” Dornburg says. “How equipped are other species of conservation concern for a battle with an emergent pathogen?”
“By understanding the genetic diversity of the immune system we can inform captive breeding to increase the chance of a species to resist disease in the wild,” Yoder adds. “Studies like this one are invaluable for captive breeding practices going forward.”
The timing of differentiation and potency of CD8 effector function is set by RNA binding proteins
by Georg Petkau, Twm J. Mitchell, Krishnendu Chakraborty, Sarah E. Bell, Vanessa D´Angeli, Louise Matheson, David J. Turner, Alexander Saveliev, Ozge Gizlenci, Fiamma Salerno, Peter D. Katsikis, Martin Turner in Nature Communications
Scientists at the Babraham Institute have shown that two RNA binding proteins hold the key to a stronger immune response to influenza in mice. Their findings reveal that the absence of these proteins changes the potency of T cells that arise at the start on an infection. Further research could lead to implications for therapies that harness the immune system, and for vaccine design.
Researchers from the Turner lab focussed on the activity of the RNA binding proteins ZFP36 and ZFP36L1. By studying mice lacking these RNA binding proteins, the researchers were able to show that their absence in T cells during the initial phase of a viral infection leads to a superior cytotoxic immune response. When the researchers infected mice with influenza, those lacking the RNA binding proteins in T cells showed signs of fighting the infection more successfully than those with the proteins present. They also transferred cells that lacked ZFP36 and ZFP36L1 into normal mice and found that even small numbers of transferred T cells provided the same advantage when fighting an influenza infection.
Their results were surprising, explains Dr Georg Petkau, a postdoctoral researcher who led the work “One striking observation of our study is that although the absence of RNA binding proteins in T cells results in stable accelerated differentiation and enhanced cytotoxicity, this does not lead to signs of disease or tissue damage, which is often a logical consequence of overt cytotoxicity during an immune response.”
The researchers speculate that the lack of negative knock on effects could be due to accelerated viral clearance and could be explained by a faster resolution of infection in young mice. It would be interesting to see whether upon recurrent infections a large accumulation of memory cells which show enhanced cytotoxicity in absence of RNA binding proteins would become potentially dangerous with age. Understanding how these RNA binding proteins limit T cell activation may thus also have implications for autoimmune disease formation in aged individuals.
The priming of the immune response once a pathogen is detected is a critical step which significantly changes the course of an immune response; it is the point at which immune cells decide to adjust the quality and duration of the immune response to a threat. In a sense the T cells in this study have to choose their weapons before they start to battle the infection and this choice is made by RNA binding proteins. By understanding more about how the immune system processes information within hours of infection and how RNA binding proteins integrate signals to activate T cells, the researchers hope to inform how we approach vaccine design and cell therapies.
“Going forward we want to investigate how the absence of RNA binding proteins affects the formation of immune memory and whether the enhanced cytotoxicity acquired early in the response is imprinted and maintained in the memory phase.” explained Dr Martin Turner, head of the Immunology research programme. Therefore, the researchers will seek to explain their findings by investigating how the stable cytotoxic program is established early after T cell activation.
A fungal extracellular effector inactivates plant polygalacturonase-inhibiting protein
by Wei Wei, Liangsheng Xu, Hao Peng, Wenjun Zhu, Kiwamu Tanaka, Jiasen Cheng, Karen A. Sanguinet, George Vandemark, Weidong Chen in Nature Communications
A protein that allows the fungus which causes white mold stem rot in more than 600 plant species to overcome plant defenses has been identified by a team of U.S. Department of Agriculture Agricultural Research Service and Washington State University scientists.
Knowledge of this protein, called SsPINE1, could help researchers develop a new, more precise system of control measures for the Sclerotinia sclerotiorum fungus, which attacks potatoes, soybeans, sunflowers, peas, lentils, canola, and many other broad leaf crops. The damage can add up to billions of dollars in a year of bad outbreaks. S. sclerotiorum fungi cause plants to rot and die by secreting chemicals called polygalacturonases (PG), which break down the plant’s cell walls. Plants evolved to protect themselves by producing a protein that stops or inhibits the fungus’ PG, labeled PGIP, which was discovered in 1971. Since then, scientists have known that some fungal pathogens have a way to overcome plant’s PGIP. But they had not been able to identify it.
“What you have is essentially a continuous arms race between fungal pathogens and their plant hosts, an intense battle of attack, counterattack and counter-counterattack in which each is constantly developing and shifting its chemical tactics in order to bypass or overcome the other’s defenses,” said research plant pathologist Weidong Chen with the ARS Grain Legume Genetics Physiology Research Unit in Pullman, Washington, and leader of the study. The key to identifying SsPINE1 was looking outside the fungi cells, according to Chen.
“We found it by looking at the materials excreted by the fungus,” he said. “And there it was. When we found this protein, SsPINE1, which interacted with PGIP, it made sense.”
Then to prove that the protein SsPINE1 was what allowed Sclerotinia to bypass plants’ PGIP, Chen and his colleagues deleted the protein in the fungus in the lab, which dramatically reduced its impact.
“I got goosebumps when we found this protein,” said Kiwamu Tanaka, an associate professor in Washington State University’s Department of Plant Pathology and a co-author on the paper. “It answered all these questions scientists have had for the last 50 years: Why these fungi always overcome plant defenses? Why do they have such a broad host range, and why are they so successful?”
The discovery of SsPINE1 has opened new avenues to investigate for controlling white mold stem rot pathogens, including possibly even more effective, more targeted breeding to make plants naturally resistant to sclerotinia diseases. And the team has showed that other related fungal pathogens use this counter-strategy, which only serves to make this discovery even more important.
Noncanonical imprinting sustains embryonic development and restrains placental overgrowth
by Shogo Matoba, Chisayo Kozuka, Kento Miura, Kimiko Inoue, Mami Kumon, Ryoya Hayashi, Tatsuya Ohhata, Atsuo Ogura, and Azusa Inoue in Genes and Development
Researchers led by Azusa Inoue at the RIKEN Center for Integrative Medical Sciences (IMS) in Japan have discovered a gene responsible for prenatal death when critical transgenerational instructions are missing from egg cells. The study shows that in mice, failed epigenetic suppression of an X-chromosome gene called Xist leads to miscarriage and developmental abnormalities.
“This study identified genes critical for fetal development whose expression is controlled by histone modifications transmitted from eggs to the next generation,” says Inoue. “The findings have implications for understanding infertility and developing treatments.”
For embryos to develop normally, egg and sperm cells need to receive important biological instructions before they meet up. Once an egg is fertilized, some of these instructions tell genes to be turned on or off depending on whether they came from the mother or father. This process is called genomic imprinting and is the focus of the new study.
When modifications in gene expression are passed on to the next generation, they are called transgenerational epigenetic changes because they’re inheritable changes even though the DNA code remains unchanged. Inoue and his team have been studying a specific set of transgenerational epigenetic instructions given to egg cells called histone H3 lysine 27 (H3K27) trimethylation. In previous studies, they found that preventing these instructions led to prenatal death, particularly for male embryos, and also to enlarged placentas in the mothers. The new study asked whether those outcomes were directly related to failed imprinting.
The study began by knocking out a gene required for H3K27 trimethylation in eggs so that the transgenerational instructions could not be given. Next, the team added a knockout of the Xist gene to these eggs. Because the male offspring tended to die, the researchers suspected that the culprit was a gene on the sex chromosome. As it turns out, there are nine maternal genes known to be suppressed in embryos in favor of the ones with paternal origins. And only one, Xist, is on the X-chromosome.
The results were almost as expected. Prenatal death was greatly reduced, and the male-skewed lethality was gone after knocking out Xist. This showed that failed Xist imprinting was the reason for the prenatal death. However, the placenta was still enlarged. Reasoning that this was likely related excess expression of the other eight genes that failed to imprint, the team created eight different deletion mutants in the double knockout embryos. They found that for three of the genes, this resulted in normal-sized placentas.
“We succeeded in curing developmental defects in a mouse model that otherwise suffers from prenatal lethality and placental malformation due to the lack of transgenerational epigenetic instructions from mothers,” says Inoue. The researchers plan to conduct more experiments to determine how these specific biological instructions are established when egg cells are created, and whether environmental factors can influence the process.
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