The discovery is unusual because the enzymes do not bear a resemblance—in their structures or amino-acid sequences—to any known class of enzymes. The team of scientists nevertheless identified them as "outlier" members of the serine/threonine hydrolase class, using newer techniques that detect biochemical activity. "A huge fraction of the human 'proteome' remains uncharacterized, and this paper shows how chemical approaches can be used to uncover proteins of a given functionality that have eluded classification based on sequence or predicted structure," said co-senior author Benjamin F. Cravatt, chair of TSRI's Department of Chemical Physiology. "In this study, we found two genes that control levels of lipids with anti-diabetic and anti-inflammatory activity, suggesting exciting targets for diabetes and inflammatory diseases," said co-senior author Alan Saghatelian, who holds the Dr. Frederik Paulsen Chair at the Salk Institute. The study, which appears as a Nature Chemical Biology Advance Online Publication on March 28, 2016, began as an effort in the Cravatt laboratory to discover and characterize new serine/threonine hydrolases using fluorophosphonate (FP) probes—molecules that selectively bind and, in effect, label the active sites of these enzymes. Pulling FP-binding proteins out of the entire proteome of test cells and identifying them using mass spectrometry techniques, the team matched nearly all to known hydrolases. The major outlier was a protein called androgen-induced gene 1 protein (AIG1). The only other one was a distant cousin in terms of sequence, a protein called ADTRP. "Neither of these proteins had been characterized as an enzyme; in fact, there had been little functional characterization of them at all," said William H. Parsons, a research associate in the Cravatt laboratory who was co-first author of the study. Experiments on AIG1 and ADTRP revealed that they do their enzymatic work in a unique way. "It looks like they have an active site that is novel—it had never been described in the literature," said Parsons. Initial tests with panels of different enzyme inhibitors showed that AIG1 and ADTRP are moderately inhibited by inhibitors of lipases—enzymes that break down fats and other lipids. But on what specific lipids do these newly discovered outlier enzymes normally work? At the Salk Institute, the Saghatelian laboratory was investigating a class of lipids it had discovered in 2014. Known as fatty acid esters of hydroxy fatty acids (FAHFAs), these molecules showed strong therapeutic potential. Saghatelian and his colleagues had found that boosting the levels of one key FAHFA lipid normalizes glucose levels in diabetic mice and also reduces inflammation. "Ben's lab was screening panels of lipids to find the ones that their new enzymes work on," said Saghatelian, who is a former research associate in the Cravatt laboratory. "We suggested they throw FAHFAs in there—and these turned out to be very good substrates." The Cravatt laboratory soon developed powerful inhibitors of the newly discovered enzymes, and the two labs began working together, using the inhibitors and genetic techniques to explore the enzymes' functions in vitro and in cultured cells. Co-first author Matthew J. Kolar, an MD-PhD student, performed most of the experiments in the Saghatelian lab. The team concluded that AIG1 and ADTRP, at least in the cell types tested, appear to work mainly to break down FAHFAs and not any other major class of lipid. In principle, inhibitors of AIG1 and ADTRP could be developed into FAHFA-boosting therapies. "Our prediction," said Saghatelian, "is that if FAHFAs do what we think they're doing, then using an enzyme inhibitor to block their degradation would make FAHFA levels go up and should thus reduce inflammation as well as improve glucose levels and insulin sensitivity." The two labs are now collaborating on further studies of the new enzymes—and the potential benefits of inhibiting them—in mouse models of diabetes, inflammation and autoimmune disease. "One of the neat things this study shows," said Cravatt, "is that even for enzyme classes as well studied as the hydrolases, there may still be hidden members that, presumably by convergent evolution, arrived at that basic enzyme mechanism despite sharing no sequence or structural homology." Explore further: New class of compounds offers great potential for research and drug development More information: AIG1 and ADTRP are atypical integral membrane hydrolases that degrade bioactive FAHFAs, Nature Chemical Biology, DOI: 10.1038/nchembio.2051
News Article | April 27, 2016
For the first time, scientists at The Scripps Research Institute (TSRI) have solved the structure of the biological machinery used by a common virus to recognize and attack human host cells. The new structure gives scientists the first view of the glycoprotein of lymphocytic choriomeningitis virus (LCMV), a virus present on every continent except Antarctica. Not only does the research reveal important traits in LCMV, it also points to possible drug targets on LCMV’s close relative: Lassa virus. “LCMV has been a beacon that has illuminated immunology and virology for decades,” said TSRI Professor Erica Ollmann Saphire. “This structure provides the missing roadmap to understand how to defend against its extremely lethal cousin, Lassa virus.” The study was published April 25, 2016, in the journal Nature Structural & Molecular Biology. LCMV is a rodent-borne virus that rarely causes noticeable symptoms in people, although it can progress to cause dangerous swelling in the brain and spinal cord of immunocompromised patients and birth defects when contracted during pregnancy. Over the years, the virus has served as a tool for understanding how the body responds to viral infections. “LCMV was the experimental virus that has illuminated much of what we understand about immunology and virology,” said Saphire. These studies included work in Frank Dixon’s lab at the fledgling Scripps Clinic and Research Foundation. Saphire describes LCMV as a “beacon” for virology. TSRI Professor Michael Oldstone, co-author of the new study, did much of that foundational work on LCMV and has long recognized LCMV’s similarity to Lassa virus, which looks identical under an electron microscope and shares 65 percent identity in the glycoprotein gene. Lassa is a much more deadly disease, however, causing thousands of deaths every year. While LCMV is a critical research tool, in the past 80 years scientists have been missing an important piece of the puzzle: a structural understanding of the proteins LCMV uses to initiate infection. Solving that structure took 10 years and has led to interesting insights into members of the arenavirus family—and how to stop them. For the study, researchers in Saphire’s lab used a method called X-ray crystallography to build three-dimensional models of the viral machinery, called the surface glycoprotein, which LCMV uses to fuse with host cells. Building the models required the scientists to screen hundreds of crystals until they found one that was stable enough to yield the necessary data. When the team finally solved the structure, it showed that the surface glycoprotein is made up of a two-part “dimer.” The dimer is made of two identical complexes that point opposite directions—“It’s like a yin and yang,” said Saphire. Two protein subunits make up each complex. One subunit, termed GP1, attaches onto the cell to be infected. The other subunit, called GP2, serves as the infection machinery, launching the process by which the virus fuses into the cell and hijacks it for its own purposes. The researchers compared each complex to an ice cream cone: GP1 forms the scoop of ice cream and GP2 forms a cone that cradles the scoop. Then there’s a long drip (the N-terminal strand of GP1) running down the side of the cone. “This structure is extremely important scientifically because it’s the first pre-fusion structure for any arenavirus glycoprotein,” said TSRI Senior Research Associate Kathryn Hastie, who was co-first author of the study with Sébastien Igonet, formerly of TSRI, now at CALIXAR. The new view of the virus came with some big surprises. For one thing, the dimer suggested that LCMV’s structure is a sort of “missing link” between two classes of viruses. Class I viruses, such as HIV, have a three-part “trimer” structure forming their fusion machinery while class II viruses, such as dengue, have a more rounded protein coat covering the whole virus. LCMV’s dimer lies flat, like class II proteins, but it likely brings in a third subunit later, creating a class I-like trimer. “It has moving parts,” explained Saphire. She added that LCMV now looks like it sits between class I and class II viruses, making this structure a possible “fossil” of an intermediate evolutionary process. In work spearheaded by TSRI biologist Brian Sullivan, the researchers studied how LCMV assembles and interacts with cells. By adding individual mutations to the genes for LCMV’s surface glycoprotein, the researchers identified five protein “residues” necessary for the virus to bind to host cells. These experiments also showed that, although the dimer is just a stage in the LCMV life cycle, it is a particularly critical stage. Disruption of the dimeric structure prevents viral growth. What This Means for Lassa Virus The researchers said these findings shed light on how this viral machine moves and rotates during infection, an action that could be blocked by drugs and antibodies. The new study also raises questions as to the degree of similarity between Lassa and LCMV. Could the dimer structure be particular to LCMV or is it a shared feature? “That information, and the fold of the GP1-GP2 complex, will be critical in the design of antibody cocktails for the treatment of Lassa fever,” said Hastie.
The relief valve, known as VRAC (volume-regulated anion channel), normally keeps cells from taking in too much water and swelling excessively. But VRAC's importance to cellular health is just beginning to be understood—already it has been tentatively linked to stroke-induced brain damage, diabetes, immune deficiency and even cancer treatment resistance. In a study published in Cell on January 28, 2016, the scientists showed that VRAC is a complex structure with five different protein subunits—the precise mix of which determines its relief-valve properties. The team also determined that VRAC's relief-valve function is activated not by the physical swelling of a cell per se, but by a closely linked event: the low concentration of dissolved ions that results from a sudden flow of water into a cell. "Knowing how VRAC is assembled and how it works is important not only because it is a fundamental regulatory mechanism in cells, but also because it seems to have relevance for a variety of diseases and conditions," said principal investigator Ardem Patapoutian, a professor at TSRI and a Howard Hughes Medical Institute (HHMI) Investigator. Scientists discovered VRAC's existence decades ago, but only recently began to identify its components. In early 2014, Patapoutian's laboratory and a separate group in Germany discovered independently that one VRAC subunit is a protein called LRRC8A (SWELL1), which is necessary for VRAC to function properly. It was apparent at the time, however, that VRAC has other subunits. In the new study, Patapoutian and his team sought a more complete understanding of how VRAC is put together and how it senses volume changes. Knowing that LRRC8A is always present in VRAC, co-first author Zhaozhu Qiu, a postdoctoral fellow in the Patapoutian lab and at the Genomics Institute of the Novartis Research Foundation (GNF), created test cells that produce LRRC8A with a special protein tag attached. The tagged LRRC8A were used as a handle to pull the full VRAC complex out of the molecular soup contained in cells. Co-first author Ruhma Syeda, also a postdoctoral fellow in the Patapoutian lab, led the effort to put purified VRAC complexes into model cell membranes (lipid bilayers) to measure the conductance of charged ions. The results were startling. Although an ion channel typically has a sharply defined single channel conductance, measurements of VRAC suggested a broad range of conductances. Prior research had suggested that the VRAC structure can include other members of the LRRC8 family besides LRRC8A, namely LRRC8B, LRRC8C, LRRC8D and/or LRRC8E. Qiu and Stuart Cahalan, another postdoctoral fellow, therefore created a set of cell lines in which genes for one or more of the LRRC8 proteins were deleted. With this and other methods, the team established that VRAC is in fact a diverse family of ion channels, each of which has approximately six protein subunits. At least one subunit of any VRAC structure is LRRC8A, but the other subunits appear to be a variable mix of LRRC8B-E proteins. That variability of composition leads to different charge-flow properties when channel complexes were measured in the minimalistic bilayer system. Swetha Murthy, a postdoctoral fellow, and Adrienne Dubin, an assistant professor of neuroscience (and co-corresponding author), determined that charge-flow properties of single VRAC channels on intact swollen cells were also dictated by the subunit combination. "We speculate that different cell types need different forms of VRAC to cope with their different environments—that's an idea we're keen to test," said Qiu. "This finding also suggests that subtle variations in VRAC's composition can have profound effects on how it works in cells and potentially contributes to disease," said Patapoutian. Perhaps the biggest unanswered question about VRAC has been: how does it sense the swelling of a cell? "People have scratched their heads over this for decades, because it's hard to imagine how a cell could directly measure an increase in its volume," Patapoutian said. One possibility has been that VRAC senses a volume increase indirectly, by detecting the stretching of the cell membrane, as some sensory ion channels do. However, examining VRAC complexes in the simplified environment of lipid bilayers, the team found that they were not activated by membrane stretching. They were, however, readily activated when the usual concentration of dissolved ions was reduced. That made sense. "Local decrease in the ionic strength is an inevitable result when the water rushes in and the cell swells," Syeda said. Further studies of VRAC will be aimed at determining its precise physical structure, how variations in that structure alter its ion-conducting properties, how VRAC varies in different cell types, and how VRAC variants or mutants contribute to disease. VRAC is thought to worsen stroke-related brain damage and heart-attack damage, for example, by allowing abnormal, harmful flows of signaling molecules in the low-oxygen condition following arterial blockage. VRAC may also be linked to immune system development: a 2003 study found that a mutation of LRRC8A, now known to be VRAC's chief subunit, prevents antibody-producing B cells from developing normally. A more recent study implicated VRAC in the clinical response to the cancer drugs cisplatin and carboplatin—the drug molecules use VRACs as portals into tumor cells. Explore further: Team solves decades-old mystery of how cells keep from bursting
Home > Press > TSRI researchers develop versatile new way to build molecules Abstract: Chemists at The Scripps Research Institute (TSRI) have devised a new and widely applicable technique for building potential drug molecules and other organic compounds. The new method, reported in the January 15, 2016 issue of the journal Science, enables researchers to add clusters of atoms called carbon fragment or functional groups to certain organic molecules more efficiently, robustly and selectively than current methods typically allow. It thus opens up new possibilities for chemists to assemble novel compounds that can be tested for useful properties in the development of drugs and other products. "We demonstrated this technique with two broad classes of compounds, aldehydes and ketones--the 'bread and butter' of modern chemical synthesis," said senior investigator Jin-Quan Yu, the Frank and Bertha Hupp Professor of Chemistry at TSRI. Expanding the Toolkit Yu's laboratory specializes in the development of techniques to make molecule-building easier, particularly for chemists trying to devise potential new drugs. Yu and his team have published more than a half-dozen of these innovations in Science or Nature in the past two years alone. Their newest tool improves a basic molecule-building operation called C-H functionalization. When chemists set out to build a candidate drug molecule, they often start with a simple organic compound whose central structure contains more inert carbon hydrogen bonds than reactive carbon heteroatom bonds. Turning such a starter molecule into a useful drug typically means replacing at least one of the hydrogen atoms with a more complex cluster of atoms called a functional group. This C-H functionalization process can be tricky for a variety of reasons, and chemists often have to employ special methods to make it work. Many of these methods involve helper molecules known as "directing groups." Chemists first attach a directing group to the initial molecule they want to modify; the directing group then guides a bond-breaking catalyst, often a metal such as palladium, to the carbon-hydrogen bond that needs to be broken to make way for the new functional group. "This has proven to be a very reliable and broadly useful strategy," said Yu, "but it requires at least two additional steps--the installation of the directing group and later its removal--and sometimes the directing group is incompatible with functional groups already present on the starting molecule." Ideally, chemists would like to find directing groups that are broadly tolerant of existing functional groups and that also don't have to be attached and detached in separate steps. Essentially that is what Yu and his team have achieved here. Cutting Out Two Steps The team--including co-first authors Fang-Lin Zhang, a visiting scholar from Wuhan University of Technology; Kai Hong, a postdoctoral research associate in the Yu Laboratory; and Tuan-Jie Li, a visiting scholar from Jiangsu Normal University--found that amino acid molecules (the building blocks of the proteins that help make up all known life forms) can work well as "transient directing groups" for ketone or aldehyde compounds. The amino acids attach themselves automatically to these starter compounds and remove themselves automatically after the new functional group is attached. In effect, this means that they work "catalytically," functionalizing one starter molecule after another and continually being re-used, rather than being consumed in their first reaction. This further streamlines the process and reduces the overall quantity of reagents that are needed. "In principle, all amino acids can be used as catalytic directing groups for such reactions," said Yu. "The availability of diverse amino acids makes it possible to find different reagents to suit different substrates or transformations." A further advantage of the new technique is that it can, with the proper choice of chiral amino acid directing group, preferentially generate "chiral" molecules that are functionalized just on one side. C-H functionalization reactions typically generate a roughly even mix of molecules functionalized on one side plus mirror-image molecules functionalized on the other side--yet the desirable biological activity of a drug often comes exclusively from its "right-handed" or "left-handed" chiral form. "The fact that we can do this using a simple amino acid as the directing group and ligand is phenomenal, considering the usual difficulty of such reactions," said Hong. "Essentially with this new method we're improving functionalizations by cutting out two steps in the functionalization process--by using a directing group that is catalytic, and by employing, if needed, a chiral directing group to generate chirally pure compounds," said Yu. Yu and his team are now working to extend the applicability of the new method to other broad classes of medicinal chemistry compounds such as amines and alcohols. ### The other co-author of the paper, "Functionalization of C(sp3)-H bonds using a transient directing group," was Hojoon Park of the Yu laboratory at TSRI. The work was funded in part by the National Institute of General Medical Sciences (2R01GM084019). For more information, please click If you have a comment, please us. Issuers of news releases, not 7th Wave, Inc. or Nanotechnology Now, are solely responsible for the accuracy of the content.
News Article | April 26, 2016
For the first time, scientists at The Scripps Research Institute (TSRI) have solved the structure of the biological machinery used by a common virus to recognize and attack human host cells.