CYCS

A newly evolved enzyme could one day make silicone compounds biodegradable. For the first time, scientists have engineered an enzyme that can break stubborn human-made bonds between silicon and carbon that exist in widely used chemicals known as siloxanes, or silicones. The discovery is a first step toward rendering the chemicals, which can linger in the environment, biodegradable. "Nature is an amazing chemist, and her repertoire now includes breaking bonds in siloxanes previously thought to evade attack by living organisms," says Frances Arnold, the Linus Pauling Professor of Chemical Engineering, Bioengineering and Biochemistry at Caltech and winner of the 2018 Nobel Prize in Chemistry for her pioneering work in directed evolution, a method for engineering enzymes and other proteins using the principles of artificial selection. Arnold and her colleagues, including Dimitris (Dimi) Katsoulis of Michigan-based Dow Inc. used directed evolution to create the new silicon-carbon bond-cleaving enzyme. The results are published in the January 26 issue of the journal Science. The researchers say that while practical uses for their engineered enzyme could still be a decade away or more, its development opens the possibility that siloxanes could one day be degraded biologically. "For example, natural organisms could evolve in siloxane-rich environments to catalyze a similar reaction, or further improved versions of laboratory-evolved enzymes such as this one could possibly be used to treat siloxane contaminants in wastewater," Arnold says. Katsoulis explains that nature doesn't use silicon-carbon bonds, "but we do and have been for about 80 years. The volatile nature of some of these compounds warrants health and environmental research to properly understand the degradation mechanisms of these materials in the environment." Siloxane chemicals can be found in countless products, including those used in household cleaning, personal care, and the automotive, construction, electronics, and aerospace industries. The compounds' chemical backbone is made of silicon-oxygen bonds, while carbon-containing groups, often methyl, are attached to the silicon atoms. "The silicon-oxygen backbone gives the polymer an inorganic-like character while the silicon-methyl groups give the polymer organic-like characteristics. Thus, these polymers have unique material properties, such as high thermal and oxidative stability, low surface tension, and high backbone flexibility among others," Katsoulis says. Siloxanes are believed to persist in the environment for days to months, and, therefore, ongoing research aims to provide greater scientific understanding of the health and environmental safety of silicone materials. The chemicals naturally start to fragment into smaller pieces, especially in soil or aquatic environments, and those fragments become volatile or escape into the air, where they undergo degradation by reacting with free radicals in the atmosphere. Of all the bonds in siloxanes, the silicon-carbon bonds are the slowest to break down. Katsoulis approached Arnold to collaborate on efforts to speed up siloxane degradation after he read about her lab's work in coaxing nature to produce silicon-carbon bonds. In 2016, Arnold and her colleagues used directed evolution to engineer a bacterial protein called cytochrome c to form silicon-carbon bonds, a process that does not occur in nature. "We decided to get nature to do what only chemists could do -- only better," Arnold said in a Caltech news release. The research demonstrated that biology could make these bonds in ways that are more environmentally friendly than those traditionally used by chemists. In the new study, the researchers wanted to find ways to break the bonds rather than create them. The scientists used directed evolution to evolve a bacterial enzyme called cytochrome P450. Directed evolution is similar to breeding dogs or horses in that the process is designed to bring out desired traits. The researchers first identified a variant of cytochrome P450 in their collection of enzymes that had a very weak ability to break silicon-carbon bonds in so-called linear and cyclic volatile methylsiloxanes, a common subgroup of the siloxane family. They mutated the DNA of the cytochrome P450 and tested the new variant enzymes. The best performers were then mutated again, and the testing was repeated until the enzyme was active enough to enable the researchers to identify the products of the reaction and study the mechanism by which the enzyme works. "Evolving enzymes to break these bonds in siloxanes presented unique hurdles. With directed evolution, we must evaluate hundreds of new enzymes in parallel to identify a few enzyme variants with improved activity," says Tyler Fulton (PhD '22), co-lead author of the study and a postdoctoral scholar at Caltech in Arnold's lab. One challenge involved the siloxane molecules leaching plastic components from the 96-well plates used to screen the variants. To solve the problem, the team created new plates made from common lab supplies. "Another challenge was finding the starting enzyme for the directed evolution process, one with even just a tiny amount of the desired activity," Arnold says. "We found it in our unique collection of cytochrome P450s evolved in the laboratory for other types of new-to-nature silicon chemistry." The final improved enzyme does not directly cleave the silicon-carbon bond but rather oxidizes a methyl group in the siloxanes in two sequential steps. Basically, this means that two carbon-hydrogen bonds are replaced with carbon-oxygen bonds, and this change allows the silicon-carbon bond to break more readily. The research draws parallels to studies involving a plastic-eating enzyme, explains Fulton, referring to a polyethylene terephthalate (PET)-degrading enzyme discovered in the bacteria Ideonella sakaiensis in 2016 by a different group of researchers. "While the PET-degrading enzyme was discovered by nature rather than by engineers, that enzyme inspired other innovations that are finally coming to fruition for plastic degradation. We hope this demonstration will similarly inspire further work to help break down siloxane compounds," he says.

Scientists have discovered how some cancer cells survive treatment and cause cancer to recur, along with a potential way to stop the process. Scientists at St. Jude Children's Research Hospital have identified how some cancer cells cheat treatment-induced cell death. In doing so, they persist and lead to cancer recurrence. The findings may serve as the basis for drugs that prevent relapses by inhibiting cancer cells from gaining these persistence traits. The research was published today in Cell. After treatment, sometimes the cancer returns, called a recurrence. Researchers knew that a small population of cancer cells sometimes become drug resistant and persist after treatment. These "persister" cells can then reconstitute a more aggressive form of the same cancer. Until now it was unclear how these cells initially change to become persistent. "When it comes to cancer cells, what doesn't kill them makes them stronger," said corresponding author Doug Green, Ph.D., Department of Immunology chair. "The field has begun to recognize that just because a cell engages apoptosis [a cell death pathway] doesn't mean it will die. Our conceptual leap was that such 'near death experiences' could be responsible for the generation of persister cells. This was unexpected -- it was like finding a piece of a treasure map that you never knew was missing -- new paths to discovery have opened up." A near-death experience Many drugs to treat cancer trigger apoptosis. St. Jude researchers found that a key event that leads to apoptosis, the release of the protein cytochrome c from mitochondria, occurs in persister cells. Historically, researchers believed that once cytochrome c was released into the cell, apoptosis could not be stopped. The evidence that some cells survive the process has grown, but it was unclear how, or why survival would lead to more aggressive cancer. The St. Jude group showed in the lab that these persistor cells do start apoptosis and that this near-death experience is key to their survival. Investigators showed that cytochrome c release kickstarts another process that can override the cell death pathway. In this study, the scientists found that when cytochrome c is released, persister cells activate a pathway called the integrated stress response in addition to apoptosis. The stress response pathway is normally used by cells to detect a problem and fix it. In persister cells, the stress response pathway stops apoptosis and promotes the expression of genes that prolong survival. "The phenomenon of persistence is caused by the 'near death experience' of engaging the mitochondrial pathway of apoptosis without dying," Green said. "We found that the generation of persister cells requires the process that leads to cytochrome c release, but instead of undergoing apoptosis, the cells survive and become persisters. In effect, these are cells that have undergone 'failed apoptosis.'" The simultaneous promotion of survival and inhibition of apoptosis also may explain why persister cells become resistant to other treatments in addition to the original drug used to treat the cancer. Though such treatments do have different mechanisms of action, most drugs ultimately induce apoptosis. Because apoptosis is inhibited, these persister cell have a general resistance to cancer therapies. Potential drug targets The research can serve as a basis for drugs that may prevent cancer recurrence by interfering with the key protein in the stress response. In persister cells, the stress response results in an increase of the protein activating transcription factor 4 (ATF4) within the cell. ATF4 is a master regulator of the stress response, resulting in the elimination of proteins that promote cell death and the upregulation of genes that promote survival. The change in gene expression due to ATF4 appears to be critical to persister cells. If ATF4 is knocked out or inhibited, the cancer cells are unable to resist the initial cancer treatment. The same happens when the protein that activates ATF4, Heme-regulated Inhibitor (HRI), is removed or inhibited. The team discovered that genes that are regulated by ATF4 in their persister cells were similarly regulated in cancer cells from patients that had survived chemotherapy, suggesting that the process occurs during cancer treatment. A model of persister formation The authors propose a model of how persisters form. When a cancer is subjected to a pro-apoptotic drug, cytochrome c is released into the cell. This begins the process of apoptosis. Simultaneously, the protein HRI is activated by cytochrome c, as part of the stress response pathway. HRI in turn causes more ATF4 to be expressed. ATF4 then changes the cells state from dying to survival. The researchers were able to show that their artificially induced persister cells were more aggressive and formed more colonies in mouse models than the original cancer cells, matching the metastatic nature of recurrent tumors. The study was supported by grants from by the National Cancer Institute (R50CA211481, P30CA021765, R35CA231620), German Research Foundation (DFG, KA 4830/1-1), and ALSAC, the fundraising and awareness organization of St. Jude.