Oral squamous cell carcinoma (OSCC) is one of the most painful cancers, which interferes with orofacial function including talking and eating. We report that legumain (Lgmn) cleaves protease-activated receptor-2 (PAR 2 ) in the acidic OSCC microenvironment to cause pain. Lgmn is a cysteine protease of late endosomes and lysosomes that can be secreted; it exhibits maximal activity in acidic environments. The role of Lgmn in PAR 2 -dependent cancer pain is unknown. We studied Lgmn activation in human oral cancers and oral cancer mouse models. Lgmn was activated in OSCC patient tumors, compared with matched normal oral tissue. After intraplantar, facial or lingual injection, Lgmn evoked nociception in wild-type (WT) female mice but not in female mice lacking PAR 2 in Na V 1.8-positive neurons (Par 2 Na v 1.8), nor in female mice treated with a Lgmn inhibitor, LI-1. Inoculation of an OSCC cell line caused mechanical and thermal hyperalgesia that was reversed by LI-1. Par 2 Na v 1.8 and Lgmn deletion attenuated mechanical allodynia in female mice with carcinogen-induced OSCC. Lgmn caused PAR 2 -dependent hyperexcitability of trigeminal neurons from WT female mice. Par 2 deletion, LI-1, and inhibitors of adenylyl cyclase or protein kinase A (PKA) prevented the effects of Lgmn. Under acidified conditions, Lgmn cleaved within the extracellular N terminus of PAR 2 at Asn 30 ;Arg 31 , proximal to the canonical trypsin activation site. Lgmn activated PAR 2 by biased mechanisms in HEK293 cells to induce Ca 21 mobilization, cAMP formation, and PKA/protein kinase D (PKD) activation, but not b-arrestin recruitment or PAR 2 endocytosis. Thus, in the acidified OSCC microenvironment, Lgmn activates PAR 2 by biased mechanisms that evoke cancer pain.

Ozonide antimalarials, OZ277 (arterolane) and OZ439 (artefenomel), are synthetic peroxide-based antimalarials with potent activity against the deadliest malaria parasite, Plasmodium falciparum. Here we used a "multi-omics" workflow, in combination with activity-based protein profiling (ABPP), to demonstrate that peroxide antimalarials initially target the haemoglobin (Hb) digestion pathway to kill malaria parasites. Time-dependent metabolomic profiling of ozonide-treated P. falciparum infected red blood cells revealed a rapid depletion of short Hb-derived peptides followed by subsequent alterations in lipid and nucleotide metabolism, while untargeted peptidomics showed accumulation of longer Hb-derived peptides. Quantitative proteomics and ABPP assays demonstrated that Hb-digesting proteases were increased in abundance and activity following treatment, respectively. Ozonideinduced depletion of short Hb-derived peptides was less extensive in a drug-treated K13mutant artemisinin resistant parasite line (Cam3.II R539T) than in the drug-treated isogenic sensitive strain (Cam3.II rev), further confirming the association between ozonide activity and Hb catabolism. To demonstrate that compromised Hb catabolism may be a primary mechanism involved in ozonide antimalarial activity, we showed that parasites forced to rely solely on Hb digestion for amino acids became hypersensitive to short ozonide exposures. Quantitative proteomics analysis also revealed parasite proteins involved in translation and the ubiquitin-proteasome system were enriched following drug treatment, suggestive of the parasite engaging a stress response to mitigate ozonide-induced damage. Taken together, these data point to a mechanism of action involving initial impairment of Hb catabolism, and indicate that the parasite regulates protein turnover to manage ozonide-induced damage.

Neutrophil elastase is a serine protease that has been implicated in the pathogenesis of inflammatory bowel disease. Due to post-translational control of its activation and high expression of its inhibitors in the gut, measurements of total expression poorly reflect the pool of active, functional neutrophil elastase. Fluorogenic substrate probes have been used to measure neutrophil elastase activity, though these tools lack specificity and traceability. PK105 is a recently described fluorescent activity-based probe, which binds to neutrophil elastase in an activity-dependent manner. The irreversible nature of this probe allows for accurate identification of its targets in complex protein mixtures. We describe the reactivity profile of PK105b, a new analogue of PK105, against recombinant serine proteases and in tissue extracts from healthy mice and from models of inflammation induced by oral cancer and Legionella pneumophila infection. We apply PK105b to measure neutrophil elastase activation in an acute model of experimental colitis. Neutrophil elastase activity is detected in inflamed, but not healthy, colons. We corroborate this finding in mucosal biopsies from patients with ulcerative colitis. Thus, PK105b facilitates detection of neutrophil elastase activity in tissue lysates, and we have applied it to demonstrate that this protease is unequivocally activated during colitis.