Functional drug screening reveals anticonvulsants as enhancers of mTOR-independent autophagic killing of Mycobacterium tuberculosis through inositol depletion.

Mycobacterium tuberculosis (MTB) remains a major challenge to global health made worse by the spread of multidrug resistance. We therefore examined whether stimulating intracellular killing of mycobacteria through pharmacological enhancement of macroautophagy might provide a novel therapeutic strategy. Despite the resistance of MTB to killing by basal autophagy, cell-based screening of FDA-approved drugs revealed two anticonvulsants, carbamazepine and valproic acid, that were able to stimulate autophagic killing of intracellular M. tuberculosis within primary human macrophages at concentrations achievable in humans. Using a zebrafish model, we show that carbamazepine can stimulate autophagy in vivo and enhance clearance of M. marinum, while in mice infected with a highly virulent multidrug-resistant MTB strain, carbamazepine treatment reduced bacterial burden, improved lung pathology and stimulated adaptive immunity. We show that carbamazepine induces antimicrobial autophagy through a novel, evolutionarily conserved, mTOR-independent pathway controlled by cellular depletion of myo-inositol. While strain-specific differences in susceptibility to in vivo carbamazepine treatment may exist, autophagy enhancement by repurposed drugs provides an easily implementable potential therapy for the treatment of multidrug-resistant mycobacterial infection

Effective Caspase Inhibition Blocks Neutrophil Apoptosis and Reveals Mcl-1 as Both a Regulator and a Target of Neutrophil Caspase Activation

Human tissue inflammation is terminated, at least in part, by the death of inflammatory neutrophils by apoptosis. The regulation of this process is therefore key to understanding and manipulating inflammation resolution. Previous data have suggested that the short-lived pro-survival Bcl-2 family protein, Mcl-1, is instrumental in determining neutrophil lifespan. However, Mcl-1 can be cleaved following caspase activity, and the possibility therefore remains that the observed fall in Mcl-1 levels is due to caspase activity downstream of caspase activation, rather than being a key event initiating apoptosis in human neutrophils

Serum and glucocorticoid-regulated kinase 1 regulates neutrophil clearance during inflammation resolution

The inflammatory response is integral to maintaining health, by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralise invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein Serum and Glucocorticoid Regulated Kinase 1 (SGK1). We have characterised the expression patterns and regulation of SGK family members in human neutrophils, and shown that inhibition of SGK activity completely abrogates the anti-apoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signalling, and thus may prove a valuable therapeutic target for the treatment of inflammatory disease

Structural control of caspase-generated glutamyl-tRNA synthetase by appended noncatalytic WHEP domains

Aminoacyl-tRNA synthetases are ubiquitous, evolutionarilyconserved enzymes catalyzing the conjugation of amino acidsonto cognate tRNAs. During eukaryotic evolution, tRNA syn-thetases have been the targets of persistent structural modifica-tions. These modifications can be additive, as in the evolution-ary acquisition of noncatalytic domains, or subtractive, as in thegeneration of truncated variants through regulated mechanismssuch as proteolytic processing, alternative splicing, or codingregion polyadenylation. A unique variant is the human glu-tamyl-prolyl-tRNA synthetase (EPRS) consisting of two fusedsynthetases joined by a linker containing three copies of theWHEP domain (termed by its presence in tryptophanyl-, histi-dyl-, and glutamyl-prolyl-tRNA synthetases). Here, we identifysite-selective proteolysis as a mechanism that severs the linkagebetween the EPRS synthetases in vitro and in vivo. Caspaseaction targeted Asp-929 in the third WHEP domain, therebyseparating the two synthetases. Using a neoepitope antibodydirected against the newly exposed C terminus, we demonstrateEPRS cleavage at Asp-929 in vitro and in vivo. Biochemical andbiophysical characterizations of the N-terminally generatedEPRS proteoform containing the glutamyl-tRNA synthetaseand most of the linker, including two WHEP domains, com-bined with structural analysis by small-angle neutron scattering,revealed a role for the WHEP domains in modulating conforma-tions of the catalytic core and GSH–S-transferase–C-terminal-like (GST-C) domain. WHEP-driven conformational rearrange-ment altered GST–C domain interactions and conferreddistinct oligomeric states in solution. Collectively, our resultsreveal long-range conformational changes imposed by theWHEP domains and illustrate how noncatalytic domains canmodulate the global structure of tRNA synthetases in complexeukaryotic systems