Protease-activated receptors in health and disease.

Proteases are signaling molecules that specifically control cellular functions by cleaving protease-activated receptors (PARs). The four known PARs are members of the large family of G protein-coupled receptors. These transmembrane receptors control most physiological and pathological processes and are the target of a large proportion of therapeutic drugs. Signaling proteases include enzymes from the circulation; from immune, inflammatory epithelial, and cancer cells; as well as from commensal and pathogenic bacteria. Advances in our understanding of the structure and function of PARs provide insights into how diverse proteases activate these receptors to regulate physiological and pathological processes in most tissues and organ systems. The realization that proteases and PARs are key mediators of disease, coupled with advances in understanding the atomic level structure of PARs and their mechanisms of signaling in subcellular microdomains, has spurred the development of antagonists, some of which have advanced to the clinic. Herein we review the discovery, structure, and function of this receptor system, highlight the contribution of PARs to homeostatic control, and discuss the potential of PAR antagonists for the treatment of major diseases.

PD-1 Inhibitory Receptor Downregulates Asparaginyl Endopeptidase and Maintains Foxp3 Transcription Factor Stability in Induced Regulatory T Cells.

CD4+ T cell differentiation into multiple T helper (Th) cell lineages is critical for optimal adaptive immune responses. This report identifies an intrinsic mechanism by which programmed death-1 receptor (PD-1) signaling imparted regulatory phenotype to Foxp3+ Th1 cells (denoted as Tbet+iTregPDL1 cells) and inducible regulatory T (iTreg) cells. Tbet+iTregPDL1 cells prevented inflammation in murine models of experimental colitis and experimental graft versus host disease (GvHD). Programmed death ligand-1 (PDL-1) binding to PD-1 imparted regulatory function to Tbet+iTregPDL1 cells and iTreg cells by specifically downregulating endo-lysosomal protease asparaginyl endopeptidase (AEP). AEP regulated Foxp3 stability and blocking AEP imparted regulatory function in Tbet+iTreg cells. Also, Aep-/- iTreg cells significantly inhibited GvHD and maintained Foxp3 expression. PD-1-mediated Foxp3 maintenance in Tbet+ Th1 cells occurred both in tumor infiltrating lymphocytes (TILs) and during chronic viral infection. Collectively, this report has identified an intrinsic function for PD-1 in maintaining Foxp3 through proteolytic pathway.

Ion Mobility-Based Enrichment-Free N-Terminomics Analysis Reveals Novel Legumain Substrates in Murine Spleen.

Aberrant levels of the asparaginyl endopeptidase legumain have been linked to inflammation, neurodegeneration, and cancer, yet our understanding of this protease is incomplete. Systematic attempts to identify legumain substrates have been previously confined to in vitro studies, which fail to mirror physiological conditions and obscure biologically relevant cleavage events. Using high-field asymmetric waveform ion mobility spectrometry (FAIMS), we developed a streamlined approach for proteome and N-terminome analyses without the need for N-termini enrichment. Compared to unfractionated proteomic analysis, we demonstrate FAIMS fractionation improves N-termini identification by >2.5 fold, resulting in the identification of >2882 unique N-termini from limited sample amounts. In murine spleens, this approach identifies 6366 proteins and 2528 unique N-termini, with 235 cleavage events enriched in WT compared to legumain-deficient spleens. Among these, 119 neo-N-termini arose from asparaginyl endopeptidase activities, representing novel putative physiological legumain substrates. The direct cleavage of selected substrates by legumain was confirmed using in vitro assays, providing support for the existence of physiologically relevant extra-lysosomal legumain activity. Combined, these data shed critical light on the functions of legumain and demonstrate the utility of FAIMS as an accessible method to improve depth and quality of N-terminomics studies.

TLR9-dependent dendritic cell maturation promotes IL-6-mediated upregulation of cathepsin X.

Cysteine cathepsins are lysosomal proteases subject to dynamic regulation within antigen-presenting cells during the immune response and associated diseases. To investigate the regulation of cathepsin X, a carboxy-mono-exopeptidase, during maturation of dendritic cells (DCs), we exposed immortalized mouse DCs to various Toll-like receptor agonists. Using a cathepsin X-selective activity-based probe, sCy5-Nle-SY, we observed a significant increase in cathepsin X activation upon TLR-9 agonism with CpG, and to a lesser extent with Pam3 (TLR1/2), FSL-1 (TLR2/6) and LPS (TLR4). Despite clear maturation of DCs in response to Poly I:C (TLR3), cathepsin X activity was only slightly increased by this agonist, suggesting differential regulation of cathepsin X downstream of TLR activation. We demonstrated that cathepsin X was upregulated at the transcriptional level in response to CpG. This occurred at late time points and was not dampened by NF-κB inhibition. Factors secreted from CpG-treated cells were able to provoke cathepsin X upregulation when applied to naïve cells. Among these factors was IL-6, which on its own was sufficient to induce transcriptional upregulation and activation of cathepsin X. IL-6 is highly secreted by DCs in response to CpG but much less so in response to poly I:C, and inhibition of the IL-6 receptor subunit glycoprotein 130 prevented CpG-mediated cathepsin X upregulation. Collectively, these results demonstrate that cathepsin X is differentially transcribed during DC maturation in response to diverse stimuli, and that secreted IL-6 is critical for its dynamic regulation.

Cathepsin X deficiency alters the processing and localisation of cathepsin L and impairs cleavage of a nuclear cathepsin L substrate.

Proteases function within sophisticated networks. Altering the activity of one protease can have sweeping effects on other proteases, leading to changes in their activity, structure, specificity, localisation, stability, and expression. Using a suite of chemical tools, we investigated the impact of cathepsin X, a lysosomal cysteine protease, on the activity and expression of other cysteine proteases and their inhibitors in dendritic cells. Among all proteases examined, cathepsin X gene deletion specifically altered cathepsin L levels; pro-cathepsin L and its single chain accumulated while the two-chain form was unchanged. This effect was recapitulated by chemical inhibition of cathepsin X, suggesting a dependence on its catalytic activity. We demonstrated that accumulation of pro- and single chain cathepsin L was not due to a lack of direct cleavage by cathepsin X or altered glycosylation, secretion, or mRNA expression but may result from changes in lysosomal oxidative stress or pH. In the absence of active cathepsin X, nuclear cathepsin L and cleavage of the known nuclear cathepsin L substrate, Lamin B1, were diminished. Thus, cathepsin X activity selectively regulates cathepsin L, which has the potential to impact the degree of cathepsin L proteolysis, the nature of substrates that it cleaves, and the location of cleavage.

MHC Class II Ubiquitination Regulates Dendritic Cell Function and Immunity.

MHC class II (MHC II) Ag presentation by dendritic cells (DCs) is critical for CD4+ T cell immunity. Cell surface levels of MHC II loaded with peptide is controlled by ubiquitination. In this study, we have examined how MHC II ubiquitination impacts immunity using MHC IIKRKI/KI mice expressing mutant MHC II molecules that are unable to be ubiquitinated. Numbers of conventional DC (cDC) 1, cDC2 and plasmacytoid DCs were significantly reduced in MHC IIKRKI/KI spleen, with the remaining MHC IIKRKI/KI DCs expressing an altered surface phenotype. Whereas Ag uptake, endosomal pH, and cathepsin protease activity were unaltered, MHC IIKRKI/KI cDC1 produced increased inflammatory cytokines and possessed defects in Ag proteolysis. Immunization of MHC IIKRKI/KI mice identified impairments in MHC II and MHC class I presentation of soluble, cell-associated and/or DC-targeted OVA via mAb specific for DC surface receptor Clec9A (anti-Clec9A-OVA mAb). Reduced T cell responses and impaired CTL killing was observed in MHC IIKRKI/KI mice following immunization with cell-associated and anti-Clec9A-OVA. Immunization of MHC IIKRKI/KI mice failed to elicit follicular Th cell responses and generated barely detectable Ab to anti-Clec9A mAb-targeted Ag. In summary, MHC II ubiquitination in DCs impacts the homeostasis, phenotype, cytokine production, and Ag proteolysis by DCs with consequences for Ag presentation and T cell and Ab-mediated immunity.

Lysosomal degradation products induce Coxiella burnetii virulence.

Coxiella burnetii is an intracellular pathogen that replicates in a lysosome-like vacuole through activation of a Dot/Icm-type IVB secretion system and subsequent translocation of effectors that remodel the host cell. Here a genome-wide small interfering RNA screen and reporter assay were used to identify host proteins required for Dot/Icm effector translocation. Significant, and independently validated, hits demonstrated the importance of multiple protein families required for endocytic trafficking of the C. burnetii-containing vacuole to the lysosome. Further analysis demonstrated that the degradative activity of the lysosome created by proteases, such as TPP1, which are transported to the lysosome by receptors, such as M6PR and LRP1, are critical for C. burnetii virulence. Indeed, the C. burnetii PmrA/B regulon, responsible for transcriptional up-regulation of genes encoding the Dot/Icm apparatus and a subset of effectors, induced expression of a virulence-associated transcriptome in response to degradative products of the lysosome. Luciferase reporter strains, and subsequent RNA-sequencing analysis, demonstrated that particular amino acids activate the C. burnetii PmrA/B two-component system. This study has further enhanced our understanding of C. burnetii pathogenesis, the host-pathogen interactions that contribute to bacterial virulence, and the different environmental triggers pathogens can sense to facilitate virulence.

Legumain Induces Oral Cancer Pain by Biased Agonism of Protease-Activated Receptor-2.

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 (PAR2) 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 PAR2-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 PAR2 in NaV1.8-positive neurons (Par2Nav1.8), 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. Par2Nav1.8 and Lgmn deletion attenuated mechanical allodynia in female mice with carcinogen-induced OSCC. Lgmn caused PAR2-dependent hyperexcitability of trigeminal neurons from WT female mice. Par2 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 PAR2 at Asn30↓Arg31, proximal to the canonical trypsin activation site. Lgmn activated PAR2 by biased mechanisms in HEK293 cells to induce Ca2+ mobilization, cAMP formation, and PKA/protein kinase D (PKD) activation, but not ß-arrestin recruitment or PAR2 endocytosis. Thus, in the acidified OSCC microenvironment, Lgmn activates PAR2 by biased mechanisms that evoke cancer pain.SIGNIFICANCE STATEMENT Oral squamous cell carcinoma (OSCC) is one of the most painful cancers. We report that legumain (Lgmn), which exhibits maximal activity in acidic environments, cleaves protease-activated receptor-2 (PAR2) on neurons to produce OSCC pain. Active Lgmn was elevated in OSCC patient tumors, compared with matched normal oral tissue. Lgmn evokes pain-like behavior through PAR2 Exposure of pain-sensing neurons to Lgmn decreased the current required to generate an action potential through PAR2 Inhibitors of adenylyl cyclase and protein kinase A (PKA) prevented the effects of Lgmn. Lgmn activated PAR2 to induce calcium mobilization, cAMP formation, and activation of protein kinase D (PKD) and PKA, but not ß-arrestin recruitment or PAR2 endocytosis. Thus, Lgmn is a biased agonist of PAR2 that evokes cancer pain.

System-wide biochemical analysis reveals ozonide antimalarials initially act by disrupting Plasmodium falciparum haemoglobin digestion.

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. Ozonide-induced depletion of short Hb-derived peptides was less extensive in a drug-treated K13-mutant artemisinin resistant parasite line (Cam3.IIR539T) than in the drug-treated isogenic sensitive strain (Cam3.IIrev), 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.

TrkB receptor cleavage by delta-secretase abolishes its phosphorylation of APP, aggravating Alzheimer's disease pathologies.

Neurotrophins promote neuronal survival and synaptic plasticity via activating the tropomyosin receptor kinases. BDNF and its high-affinity receptor TrkB are reduced in Alzheimer's disease (AD), contributing to progressive cognitive decline. However, how the signaling mediates AD pathologies remains incompletely understood. Here we show that the TrkB receptor binds and phosphorylates APP, reducing amyloid-ß production, which are abrogated by δ-secretase cleavage of TrkB in AD. Remarkably, BDNF stimulates TrkB to phosphorylate APP Y687 residue that accumulates APP in the TGN (Trans-Golgi Network) and diminishes its amyloidogenic cleavage. Delta-secretase cleaves TrkB at N365 and N486/489 residues and abolishes its neurotrophic activity, decreasing p-APP Y687 and altering its subcellular trafficking. Notably, both TrkB and APP are robustly cleaved by δ-secretase in AD brains, accompanied by mitigated TrkB signaling and reduced p-Y687. Blockade of TrkB cleavage attenuates AD pathologies in 5xFAD mice, rescuing the learning and memory. Viral expression of TrkB 1-486 fragment in the hippocampus of APP/PS1 mice facilitates amyloid pathology and mitigates cognitive functions. Hence, δ-secretase cleaves TrkB and blunts its phosphorylation of APP, facilitating AD pathogenesis.

Mitochondrial dysfunction triggers the pathogenesis of Parkinson's disease in neuronal C/EBPß transgenic mice.

Respiratory chain complex I deficiency elicits mitochondrial dysfunction and reactive oxidative species (ROS), which plays a crucial role in Parkinson's disease (PD) pathogenesis. However, it remains unclear whether the impairment in other complexes in the mitochondrial oxidative phosphorylation chain is also sufficient to trigger PD onset. Here we show that inhibition of Complex II or III in the electron transport chain (ETC) induces the motor disorder and PD pathologies in neuronal Thy1-C/EBPß transgenic mice. Through a cell-based screening of mitochondrial respiratory chain inhibitors, we identified TTFA (complex II inhibitor) and Atovaquone (complex III inhibitor), which robustly block the oxidative phosphorylation functions, strongly escalate ROS, and activate C/EBPß/AEP pathway that triggers dopaminergic neuronal cell death. Oral administration of these inhibitors to Thy1-C/EBPß mice elicits constipation and motor defects, associated with Lewy body-like inclusions. Deletion of SDHD (Succinate dehydrogenase) gene from the complex II in the Substantia Nigra of Thy1-C/EBPß mice triggers ROS and PD pathologies, resulting in motor disorders. Hence, our findings demonstrate that mitochondrial ETC inactivation triggers PD pathogenesis via activating C/EBPß/AEP pathway.

Protease-activated receptor-2 in endosomes signals persistent pain of irritable bowel syndrome.

Once activated at the surface of cells, G protein-coupled receptors (GPCRs) redistribute to endosomes, where they can continue to signal. Whether GPCRs in endosomes generate signals that contribute to human disease is unknown. We evaluated endosomal signaling of protease-activated receptor-2 (PAR2), which has been proposed to mediate pain in patients with irritable bowel syndrome (IBS). Trypsin, elastase, and cathepsin S, which are activated in the colonic mucosa of patients with IBS and in experimental animals with colitis, caused persistent PAR2-dependent hyperexcitability of nociceptors, sensitization of colonic afferent neurons to mechanical stimuli, and somatic mechanical allodynia. Inhibitors of clathrin- and dynamin-dependent endocytosis and of mitogen-activated protein kinase kinase-1 prevented trypsin-induced hyperexcitability, sensitization, and allodynia. However, they did not affect elastase- or cathepsin S-induced hyperexcitability, sensitization, or allodynia. Trypsin stimulated endocytosis of PAR2, which signaled from endosomes to activate extracellular signal-regulated kinase. Elastase and cathepsin S did not stimulate endocytosis of PAR2, which signaled from the plasma membrane to activate adenylyl cyclase. Biopsies of colonic mucosa from IBS patients released proteases that induced persistent PAR2-dependent hyperexcitability of nociceptors, and PAR2 association with ß-arrestins, which mediate endocytosis. Conjugation to cholestanol promoted delivery and retention of antagonists in endosomes containing PAR2 A cholestanol-conjugated PAR2 antagonist prevented persistent trypsin- and IBS protease-induced hyperexcitability of nociceptors. The results reveal that PAR2 signaling from endosomes underlies the persistent hyperexcitability of nociceptors that mediates chronic pain of IBS. Endosomally targeted PAR2 antagonists are potential therapies for IBS pain. GPCRs in endosomes transmit signals that contribute to human diseases.

N-Terminomics/TAILS Profiling of Macrophages after Chemical Inhibition of Legumain.

Legumain (asparaginyl endopeptidase) is the only protease with a preference for cleavage after asparagine residues. Increased legumain activity is a hallmark of inflammation, neurodegenerative diseases, and cancer, and legumain inhibitors have exhibited therapeutic effects in mouse models of these pathologies. Improved knowledge of its substrates and cellular functions is a requisite to further validation of legumain as a drug target. We, therefore, aimed to investigate the effects of legumain inhibition in macrophages using an unbiased and systematic approach. By shotgun proteomics, we identified 16 094 unique peptides in RAW264.7 cells. Among these, 326 unique peptides were upregulated in response to legumain inhibition, while 241 were downregulated. Many of these proteins were associated with mitochondria and metabolism, especially iron metabolism, indicating that legumain may have a previously unknown impact on related processes. Furthermore, we used N-terminomics/TAILS (terminal amine isotopic labeling of substrates) to identify potential substrates of legumain. We identified three new proteins that are cleaved after asparagine residues, which may reflect legumain-dependent cleavage. We confirmed that frataxin, a mitochondrial protein associated with the formation of iron-sulfur clusters, can be cleaved by legumain. This further asserts a potential contribution of legumain to mitochondrial function and iron metabolism. Lastly, we also identified a potential new cleavage site within legumain itself that may give rise to a 25 kDa form of legumain that has previously been observed in multiple cell and tissue types. Collectively, these data shed new light on the potential functions of legumain and will be critical for understanding its contribution to disease.

High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections - Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils.

BACKGROUND: Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. RESULTS: Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. CONCLUSIONS: Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.

Myoepithelial cell-specific expression of stefin A as a suppressor of early breast cancer invasion.

Mammography screening has increased the detection of early pre-invasive breast cancers, termed ductal carcinoma in situ (DCIS), increasing the urgency of identifying molecular regulators of invasion as prognostic markers to predict local relapse. Using the MMTV-PyMT breast cancer model and pharmacological protease inhibitors, we reveal that cysteine cathepsins have important roles in early-stage tumorigenesis. To characterize the cell-specific roles of cathepsins in early invasion, we developed a DCIS-like model, incorporating an immortalized myoepithelial cell line (N1ME) that restrained tumor cell invasion in 3D culture. Using this model, we identified an important myoepithelial-specific function of the cysteine cathepsin inhibitor stefin A in suppressing invasion, whereby targeted stefin A loss in N1ME cells blocked myoepithelial-induced suppression of breast cancer cell invasion. Enhanced invasion observed in 3D cultures with N1ME stefin A-low cells was reliant on cathepsin B activation, as addition of the small molecule inhibitor CA-074 rescued the DCIS-like non-invasive phenotype. Importantly, we confirmed that stefin A was indeed abundant in myoepithelial cells in breast tissue. Use of a 138-patient cohort confirmed that myoepithelial stefin A (cystatin A) is abundant in normal breast ducts and low-grade DCIS but reduced in high-grade DCIS, supporting myoepithelial stefin A as a candidate marker of lower risk of invasive relapse. We have therefore identified myoepithelial cell stefin A as a suppressor of early tumor invasion and a candidate marker to distinguish patients who are at low risk of developing invasive breast cancer, and can therefore be spared further treatment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Fluorescent diphenylphosphonate-based probes for detection of serine protease activity during inflammation.

Activity-based probes are small molecules that covalently bind to the active site of a protease in an activity-dependent manner. We synthesized and characterized two fluorescent activity-based probes that target serine proteases with trypsin-like or elastase-like activity. We assessed the selectivity and potency of these probes against recombinant enzymes and demonstrated that while they are efficacious at labeling active proteases in complex protein mixtures in vitro, they are less valuable for in vivo studies. We used these probes to evaluate serine protease activity in two mouse models of acute inflammation, including pancreatitis and colitis. As anticipated, the activity of trypsin-like proteases was increased during pancreatitis. Levels of elastase-like proteases were low in pancreatic lysates and colonic luminal fluids, whether healthy or inflamed. Exogenously added recombinant neutrophil elastase was inhibited upon incubation with these samples, an effect that was augmented in inflamed samples compared to controls. These data suggest that endogenous inhibitors and elastase-degrading proteases are upregulated during inflammation.

Pathophysiological roles of proteases in gastrointestinal disease.

Gastrointestinal diseases, such as irritable bowel syndrome, inflammatory bowel disease, and colorectal cancer, affect a large proportion of the population and are associated with many unpleasant symptoms. Although the causes of these diseases remain largely unknown, there is increasing evidence to suggest that dysregulated protease activity may be a contributing factor. Proteases are enzymes that cleave other proteins, and their activity is normally very tightly regulated. During disease, however, the balance between proteases and their inhibitors is often shifted, leading to altered spatial and temporal control of substrate cleavage. Evaluating protease levels in normal physiology and disease has relied heavily on the use of chemical tools. Although these tools have greatly advanced the field, they are not without caveats. This review provides an introduction to these tools, their application in the gut, and a summary of the current knowledge on the contribution of protease activity to gastrointestinal disease.

Legumain is activated in macrophages during pancreatitis.

Pancreatitis is an inflammatory disease of the pancreas characterized by dysregulated activity of digestive enzymes, necrosis, immune infiltration, and pain. Repeated incidence of pancreatitis is an important risk factor for pancreatic cancer. Legumain, a lysosomal cysteine protease, has been linked to inflammatory diseases such as atherosclerosis, stroke, and cancer. Until now, legumain activation has not been studied during pancreatitis. We used a fluorescently quenched activity-based probe to assess legumain activation during caerulein-induced pancreatitis in mice. We detected activated legumain by ex vivo imaging, confocal microscopy, and gel electrophoresis. Compared with healthy controls, legumain activity in the pancreas of caerulein-treated mice was increased in a time-dependent manner. Legumain was localized to CD68(+) macrophages and was not active in pancreatic acinar cells. Using a small-molecule inhibitor of legumain, we found that this protease is not essential for the initiation of pancreatitis. However, it may serve as a biomarker of disease, since patients with chronic pancreatitis show strongly increased legumain expression in macrophages. Moreover, the occurrence of legumain-expressing macrophages in regions of acinar-to-ductal metaplasia suggests that this protease may influence reprogramming events that lead to inflammation-induced pancreatic cancer.

Murine norovirus infection of macrophages induces intrinsic apoptosis as the major form of programmed cell death.

Human norovirus is the leading cause of acute gastroenteritis worldwide, however despite the significance of this pathogen, we have a limited understanding of how noroviruses cause disease, and modulate the innate immune response. Programmed cell death (PCD) is an important part of the innate response to invading pathogens, but little is known about how specific PCD pathways contribute to norovirus replication. Here, we reveal that murine norovirus (MNV) virus-induced PCD in macrophages correlates with the release of infectious virus. We subsequently show, genetically and chemically, that MNV-induced cell death and viral replication occurs independent of the activity of inflammatory mediators. Further analysis revealed that MNV infection promotes the cleavage of apoptotic caspase-3 and PARP. Correspondingly, pan-caspase inhibition, or BAX and BAK deficiency, perturbed viral replication rates and delayed virus release and cell death. These results provide new insights into how MNV harnesses cell death to increase viral burden.

Chemical Tools to Image the Activity of PAR-Cleaving Proteases.

Protease-activated receptors (PARs) comprise a family of four G protein-coupled receptors (GPCRs) that have broad functions in health and disease. Unlike most GPCRs, PARs are uniquely activated by proteolytic cleavage of their extracellular N termini. To fully understand PAR activation and function in vivo, it is critical to also study the proteases that activate them. As proteases are heavily regulated at the post-translational level, measures of total protease abundance have limited utility. Measures of protease activity are instead required to inform their function. This review will introduce several classes of chemical probes that have been developed to measure the activation of PAR-cleaving proteases. Their strengths, weaknesses, and applications will be discussed, especially as applied to image protease activity at the whole organism, tissue, and cellular level.