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Lysosomal damage activates AMPK, a regulator of macroautophagy/autophagy and metabolism, and elicits a strong ubiquitination response. Here we show that the cytosolic lectin LGALS9 detects lysosomal membrane breach by binding to lumenal glycoepitopes, and directs both the ubiquitination response and AMPK activation. Proteomic analyses have revealed increased LGALS9 association with lysosomes, and concomitant changes in LGALS9 interactions with its newly identified partners that control ubiquitination-deubiquitination processes. An LGALS9-inetractor, deubiquitinase USP9X, dissociates from damaged lysosomes upon recognition of lumenal glycans by LGALS9. USP9X's departure from lysosomes promotes K63 ubiquitination and stimulation of MAP3K7/TAK1, an upstream kinase and activator of AMPK hitherto orphaned for a precise physiological function. Ubiquitin-activated MAP3K7/TAK1 controls AMPK specifically during lysosomal injury, caused by a spectrum of membrane-damaging or -permeabilizing agents, including silica crystals, the intracellular pathogen Mycobacterium tuberculosis , TNFSF10/TRAIL signaling, and the anti-diabetes drugs metformin. The LGALS9-ubiquitin system activating AMPK represents a novel signal transduction system contributing to various physiological outputs that are under the control of AMPK, including autophagy, MTOR, lysosomal maintenance and biogenesis, immunity, defense against microbes, and metabolic reprograming., Abbreviations: AMPK: AMP-activated protein kinase; APEX2: engineered ascorbate peroxidase 2; ATG13: autophagy related 13; ATG16L1: autophagy related 16 like 1; BMMs: bone marrow-derived macrophages; CAMKK2: calcium/calmodulin dependent protein kinase kinase 2; DUB: deubiquitinase; GPN: glycyl-L-phenylalanine 2-naphthylamide; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAP3K7/TAK1: mitogen-activated protein kinase kinase kinase 7; MERIT: membrane repair, removal and replacement; MTOR: mechanistic target of rapamycin kinase; STK11/LKB1: serine/threonine kinase 11; TNFSF10/TRAIL: TNF superfamily member 10; USP9X: ubiquitin specific peptidase 9 X-linked.

Photo-oxidation of bovine serum albumin (BSA) by porphyrins produces protein-centred radicals that can be spin trapped by 3, 5-dibromo-4-nitrosobenzenesulphonic acid (DBNBS) and 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO). In the case of DMPO, a thiyl radical from the Cys-34 residue is trapped, whereas with DBNBS signals from both this thiyl and tertiary carbon-centred species are observed. However, specific chemical modification of the Cys-34 residue, in combination with dual-isotope spin-trapping techniques, shows that the signal assigned to the Cys-34 thiyl adduct with DBNBS is a nitroxide artefact resulting from sequential (non-radical) nucleophilic addition and oxidation. In contrast, both the Cys-34 thiyl DMPO adduct and the tertiary carbon-centred DBNBS adducts result from genuine spintrapping. This study shows that such artefacts can be detected-even with anisotropic EPR spectra-through the use of appropriately substituted spin-traps, and that nitroso spin-traps need to be employed with great care in systems containing free thiol groups.

EPR spin-trapping, although a powerful, sensitive technique for the study of free radicals, can be susceptible to artefacts; one of the most intractable to determine has been the non-radical addition of a substrate to a spin-trap followed by oxidation of the product to an EPR-detectable nitroxide. This work details how differentially isotopically labelled spin-traps (either nitroso or nitrone) may be used to determine the presence (or absence) of such artefacts, and provide a semi-quantitative measure of the extent of their contribution to the total EPR spectra in spin-trapping reactions. Artefactual 'ene' addition of the nitroso spin-trap 3,5-dibromo-4-nitroso-benzenesulphonic acid (DBNBS) to tryptophan followed by oxidation to EPR-detectable products has been confirmed, as has its nucleophilic addition to the thiol of glutathione to give non-EPR detectable products. The nitrone α-phenyl-N-tert-butylnitrone (PBN) exhibited no such reactivity.

We report the synthesis and use of d2-(15)N isotopically-labelled 3, 5-dibromo-4-nitrosobenzenesulphonic acid (DBNBS-d2-(15)N, as its sodium salt), a spin-trap possessing several advantages over non-labelled DBNBS. The simplification in the electron paramagnetic resonance spectra of radical adducts of DBNBS-d2-(15)N compared with those of DBNBS not only results in increased sensitivity, but also facilitates the assignment and analysis of complex spectra. An example of this simplification is given.