Blocking cGAS in Autoimmunity

October 10, 2017

Cells of the innate immune system have developed multiple sensors to detect the presence of infectious agents and certain forms of cellular damage. One such sensor is the enzyme cyclic GMP-AMP synthase (cGAS), which responds to the presence of cytosolic double-stranded DNA (dsDNA) by catalyzing the formation of a cyclic nucleotide (cGAMP) from GTP and ATP. cGAMP acts as a second messenger to activate a signaling pathway that leads to the secretion of a number of cytokines, most importantly, interferon-β1 (IFNB1). The cGAS pathway plays a role in the immune response to intracellular pathogens (viruses and bacteria), by responding to the presence of the invading organisms’ dsDNA. However, for patients bearing an inactivating mutation of TREX1, the enzyme primarily responsible for degrading cytosolic dsDNA, cGAS promotes excessive IFNB1 production, leading to an autoimmune disease similar to systemic lupus erythematosus. This led Vanderbilt Basic Sciences investigator Manuel Ascano, his collaborators Dinshaw Patel (Memorial Sloan-Kettering Cancer Center) and Fraser Glickman (The Rockefeller University), and their laboratories to search for a small molecule inhibitor of cGAS. They developed a mass spectrometry-based high-throughput screen to evaluate a library of 123,306 compounds in search of cGAS inhibitors. Then using the four most promising hits, they embarked on a structure-guided medicinal chemistry effort that produced the lead molecule RU.521. A crystal structure of RU.521 in complex with cGAS and dsDNA indicated that the inhibitor binds in the same region of the enzyme’s active site cavity as the substrates, intermediate, and product of the reaction. Kinetic studies suggested a noncompetitive inhibitory mechanism. Studies using a murine macrophage-like cell line demonstrated that the compound is active in intact cells, and that it does not interfere, directly or indirectly, with a wide range of unrelated inflammatory signaling pathways. The ability of RU.521 to reduce IFNB1 production by bone marrow-derived macrophages from Trex1 knockout mice suggested that it may be a valuable probe to investigate the role of the cGAS pathway in autoimmunity. The work is published in the journal Nature Communications [J. Vincent et al. Nat. Commun., 2013, 8:750 DOI: 10.1038/s41467-017-00833-9].

A TALK About ER Ca2+ Homeostasis

October 10, 2017

Insulin secretion by pancreatic β-cells occurs in response to glucose-induced increases in intracellular calcium (Ca2+c). Past research has shown that fluxes in Ca2+ from the endoplasmic reticulum (ER) play a role in this process, and that these fluxes can be disturbed in diabetes. The movement of Ca2+ across the ER membrane is electrically balanced by an opposing movement of K+; however, the identity of the K+ channel responsible for this movement has remained a mystery. Now, Vanderbilt Basic Sciences investigator David Jacobson and his laboratory identify the TALK-1 K+ channel as a key player in β-cell Ca2+ homeostasis. The investigators first confirmed that TALK-1 is expressed in the ER membrane of both mouse and human β-cells, and they used β-cells from TALK-1 knockout (KO) mice to show that TALK-1 decreases the amount of Ca2+ stored in the ER (Ca2+ER) while raising basal Ca2+c. These findings suggested that TALK-1 promotes leakage of Ca2+ER, and results from overexpression of the related K+ channels TASK-1 and TASK-3 in HEK293 cells indicated that they perform the same function. In fact, inhibition of TASK-1 expressed in pancreatic α-cells using a specific channel blocker reduced Ca2+ER leakage in those cells. Electrophysiology experiments confirmed that TALK-1 and TASK-1 form functional K+ channels in β- and α-cells, respectively, and they showed that Ca2+ER release is reduced in TALK-1 β-cells responding to a depolarizing stimulus. However, exposure of TALK-1 KO cells to high glucose resulted in higher Ca2+ influx and more radical Ca2+ oscillations. The investigators explained this observation by hypothesizing that release of Ca2+ER is required to stimulate a plasma membrane-based slow K+ current that dampens the oscillations. A failure of normal Ca2+ER homeostasis can lead to ER stress. The investigators found that islets from TALK-1 KO mice exhibited decreased expression of ER stress response genes in response to prolonged exposure to a high fat diet. Furthermore, cells expressing a type 2 diabetes-linked gain-of-function TALK-1 mutant showed an increased ER stress response to tunicamycin exposure. The results suggest that TALK-1 is directly related to insulin secretion and maintenance of ER homeostasis. Thus, it may be a target for drug discovery to treat inappropriate Ca2+ER release during diabetes pathogenesis. The work is published in the journal Science Signaling [N. C. Vierra, et al., (2017) Sci. Signal. 10, eaan2883].



Unique Properties of DNA Gyrase for (+) Supercoil Processing

October 2, 2017

The twisting of DNA in the same or opposite direction of the turn of the double helix gives rise to (+) or (-) supercoiling, respectively. Supercoiling exerts a strain on the helix, leading to the formation of loops, knots, and tangles that can have a major impact on the ability of DNA processing enzymes to carry out their function. This is especially true with regard to (+) supercoils that form ahead of the replication or transcription machinery as a result of DNA unwinding. In bacteria, (+) supercoils are removed through the action of two enzymes, gyrase and topoisomerase IV. Both enzymes work by grasping a strand of double-stranded DNA, introducing a break in that strand, and then passing a second strand through the break before it is reconnected. Topoisomerase IV can remove (-) as well as (+) supercoils, whereas gyrase introduces (-) supercoils in relaxed DNA. This difference in (-) supercoil processing is associated with the ability of gyrase, but not topoisomerase IV to wrap the second strand of DNA before passing it through the break. Relatively little is known, however, about how the two enzymes process (+) supercoils, leading Vanderbilt Basic Sciences investigator Neil Osheroff and his graduate student Rachel Ashley to take a closer look. They discovered that gyrase removes (+) supercoils much faster than it introduces (-) supercoils, and that it does so in bursts of activity as fast as 107 supercoils/s. A mutant enzyme unable to wrap DNA exhibited markedly reduced efficiency in (+) supercoil processing, indicating that wrapping was required for both of gyrase’s primary functions. Topoisomerase IV also removed (+) supercoils more quickly than (-) ones, but it was much less efficient than gyrase at (+) supercoil processing. When gyrase or topoisomerase IV break the first bound strand of DNA, it becomes covalently bound to the enzyme, forming a cleavage complex. Failure to rejoin the ends of the DNA strand can lead to double strand breaks and ultimately the death of the cell. Quinolone antimicrobial agents work by stabilizing the cleavage complex. The researchers showed that in the presence of quinolones, gyrase forms fewer double strand breaks while processing (+) supercoiled DNA than (-) supercoiled DNA. For topoisomerase IV, there was little difference in break formation between the two substrates. The results suggest that gyrase is particularly well-suited to process (+) supercoils formed ahead of the replication or transcription machinery in terms of both the rapidity of the process and the low rate of double strand break formation. The work is published in the journal Nucleic Acids Research [R. E. Ashley, et al. Nuc. Acids Res., 45, 9611].