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Abstract Rationale In cystic fibrosis (CF) a reduction in airway surface liquid (ASL) height compromises mucociliary clearance, favoring mucus plugging and chronic bacterial infection. Inhibitors of the epithelial sodium channel (ENaC) have therapeutic potential in CF airways to reduce hyperstimulated sodium and fluid absorption to levels that can restore airway hydration. Objectives To determine whether a novel compound (QUB-TL1) designed to inhibit protease/ENaC signaling in CF airways restores ASL volume and mucociliary function. Methods Protease activity was measured using fluorogenic activity assays. Differentiated primary airway epithelial cell cultures (F508del homozygotes) were used to determined ENaC activity (Ussing chamber recordings), ASL height (confocal microscopy), and mucociliary function (by tracking the surface flow of apically applied microbeads). Cell toxicity was measured using a lactate dehydrogenase assay. Measurements and Main Results QUB-TL1 inhibits extracellularly located channel activating proteases (CAPs), including prostasin, matriptase, and furin, the activities of which are observed at excessive levels at the apical surface of CF airway epithelial cells. QUB-TL1–mediated CAP inhibition results in diminished ENaC-mediated Na+ absorption in CF airway epithelial cells caused by internalization of a prominent pool of cleaved (active) ENaCγ from the cell surface. Importantly, diminished ENaC activity correlates with improved airway hydration status and mucociliary clearance. We further demonstrate QUB-TL1–mediated furin inhibition, which is in contrast to other serine protease inhibitors (camostat mesylate and aprotinin), affords protection against neutrophil elastase–mediated ENaC activation and Pseudomonas aeruginosa exotoxin A–induced cell death. Conclusions QUB-TL1 corrects aberrant CAP activities, providing a mechanism to delay or prevent the development of CF lung disease in a manner independent of CF transmembrane conductance regulator mutation.

Chronic obstructive pulmonary disease (COPD) is a debilitating heterogeneous disease characterised by unregulated proteolytic destruction of lung tissue mediated via a protease-antiprotease imbalance. In COPD, the relationship between the neutrophil serine protease, neutrophil elastase, and its endogenous inhibitor, alpha-1-antitrypsin (AAT) is the best characterised. AAT belongs to a superfamily of serine protease inhibitors known as serpins. Advances in screening technologies have, however, resulted in many members of the serpin superfamily being identified as having differential expression across a multitude of chronic lung diseases compared to healthy individuals. Serpins exhibit a unique suicide-substrate mechanism of inhibition during which they undergo a dramatic conformational change to a more stable form. A limitation is that this also renders them susceptible to disease-causing mutations. Identification of the extent of their physiological/pathological role in the airways would allow further expansion of knowledge regarding the complexity of protease regulation in the lung and may provide wider opportunity for their use as therapeutics to aid the management of COPD and other chronic airways diseases.

Many bacterial and viral pathogens (or their toxins), including Pseudomonas aeruginosa exotoxin A, require processing by host pro-protein convertases such as furin to cause disease. We report the development of a novel irreversible inhibitor of furin (QUB-F1) consisting of a diphenyl phosphonate electrophilic warhead coupled with a substrate-like peptide (RVKR), that also includes a biotin tag, to facilitate activity-based profiling/visualisation. QUB-F1 displays greater selectivity for furin, in comparison to a widely used exemplar compound (furin I) which has a chloromethylketone warhead coupled to RVKR, when tested against the serine trypsin-like proteases (trypsin, prostasin and matriptase), factor Xa and the cysteine protease cathepsin B. We demonstrate QUB-F1 does not prevent P. aeruginosa exotoxin A-induced airway epithelial cell toxicity; in contrast to furin I, despite inhibiting cell surface furin-like activity to a similar degree. This finding indicates additional proteases, which are sensitive to the more broad-spectrum furin I compound, may be involved in this process.

Cystic fibrosis (CF) is a life-limiting genetic disorder caused by loss-of-function mutations in the gene which codes for the CF transmembrane conductance regulator (CFTR) Cl− channel. Loss of Cl− secretion across the apical membrane of airway lining epithelial cells results in dehydration of the airway surface liquid (ASL) layer which impairs mucociliary clearance (MCC), and as a consequence promotes bacterial infection and inflammation of the airways. Interventions that restore airway hydration are known to improve MCC. Here we review the ion channels present at the luminal surface of airway epithelial cells that may be targeted to improve airway hydration and MCC in CF airways.

Trypsin-like proteases (TLPs) belong to a family of serine enzymes with primary substrate specificities for the basic residues, lysine and arginine, in the P1 position. Whilst initially perceived as soluble enzymes that are extracellularly secreted, a number of novel TLPs that are anchored in the cell membrane have since been discovered. Muco-obstructive lung diseases (MucOLDs) are characterised by the accumulation of hyper-concentrated mucus in the small airways, leading to persistent inflammation, infection and dysregulated protease activity. Although neutrophilic serine proteases, particularly neutrophil elastase, have been implicated in the propagation of inflammation and local tissue destruction, it is likely that the serine TLPs also contribute to various disease-relevant processes given the roles that a number of these enzymes play in the activation of both the epithelial sodium channel (ENaC) and protease-activated receptor 2 (PAR2). More recently, significant attention has focused on the activation of viruses such as SARS-CoV-2 by host TLPs. The purpose of this review was to highlight key TLPs linked to the activation of ENaC and PAR2 and their association with airway dehydration and inflammatory signalling pathways, respectively. The role of TLPs in viral infectivity will also be discussed in the context of the inhibition of TLP activities and the potential of these proteases as therapeutic targets.

Epithelial barrier dysfunction, characteristic of allergic airway disease may be, at least in part, due to the action of allergen-associated protease activities. Cockroach allergy is a major global health issue, with cockroaches containing considerable serine trypsin-like protease (TLP) activity. The present study sought to evaluate two novel protease inhibitors (PE-BBI and pLR-HL), recently isolated from amphibian skin secretions, for their potential to neutralise cockroach TLP activity and to determine any protective effect on cockroach-induced airway epithelial barrier disruption. Inhibitor potencies against the cockroach-associated activities were determined using a fluorogenic peptide substrate-based activity assay. 16HBE14o- cells (16HBE; a bronchial epithelial cell line) were treated with cockroach extract (CRE) in the presence or absence of the compounds in order to assess cell viability (RealTime Glo luminescent assay) and epithelial barrier disruption (transepithelial resistance and paracellular dextran flux). PE-BBI potently and selectively inhibited CRE TLP activity (pIC50 -8), but not host (16HBE) cell surface activity, which conferred protection of 16HBE cells from CRE-induced cell damage and barrier disruption. Novel protease inhibitor strategies such as PE-BBI may be useful for the treatment of allergic airway disease caused by cockroach proteases.

Background Vascular endothelial growth factors (VEGFs) are key regulators of endothelial cell function and angiogenesis. We and others have previously demonstrated that VEGF-A stimulates AMP-activated protein kinase (AMPK) in cultured endothelial cells. Furthermore, AMPK has been reported to regulate VEGF-mediated angiogenesis. The role of AMPK in the function of VEGF-B remains undetermined, as does the role of AMPK in VEGF-stimulated endothelial cell proliferation, a critical process in angiogenesis. Methods Human aortic endothelial cells (HAECs) were incubated with VEGF-A and VEGF-B prior to examination of HAEC AMPK activity, proliferation, migration, fatty acid oxidation and fatty acid transport. The role of AMPK in the functional effects of VEGF-A and/or VEGF-B was assessed after downregulation of AMPK activity with chemical inhibitors or infection with adenoviruses expressing a dominant negative mutant AMPK. Results Incubation of HAECs with VEGF-B rapidly stimulated AMPK activity in a manner sensitive to an inhibitor of Ca 2+ /calmodulin-dependent kinase kinase (CaMKK), without increasing phosphorylation of endothelial NO synthase (eNOS) phosphorylation at Ser1177. Downregulation of AMPK abrogated HAEC proliferation in response to VEGF-A or VEGF-B. However, activation of AMPK by agents other than VEGF inhibited proliferation. Downregulation of AMPK abrogated VEGF-A-stimulated HAEC migration, whereas infection with adenoviruses expressing constitutively active mutant AMPK stimulated chemokinesis. Neither VEGF-A nor VEGF-B had any significant effect on HAEC fatty acid oxidation, yet prolonged incubation with VEGF-A stimulated fatty acid uptake in an AMPK-dependent manner. Inhibition of eNOS abrogated VEGF-mediated proliferation and migration, but was without effect on VEGF-stimulated fatty acid transport, ERK or Akt phosphorylation. Conclusions These data suggest that VEGF-B stimulates AMPK by a CaMKK-dependent mechanism and stimulation of AMPK activity is required for proliferation in response to either VEGF-A or VEGF-B and migration in response to VEGF-A. AMPK activation alone was not sufficient, however, to stimulate proliferation in the absence of VEGF. VEGF-stimulated NO synthesis is required for the stimulation of proliferation by VEGF-A or VEGF-B, yet this may be independent of eNOS Ser1177 phosphorylation.

The endothelium acts to maintain vascular homeostasis, including the regulation of vascular tone, blood fluidity and coagulation. Endothelial dysfunction, a condition largely characterised by reduced NO bioavailability, is an important feature associated with the aetiology of several pathophysiological disorders including type 2 diabetes and cardiovascular disease. AMPK is the downstream component of a protein kinase cascade important in the regulation of cellular and whole body metabolism. AMPK has been demonstrated to mediate a number of physiological responses in the endothelium, including the stimulation of eNOS phosphorylation and NO synthesis; and as such AMPK represents a therapeutic target in the dysfunctional endothelium. VEGF has been established as the prime angiogenic molecule during development, adult physiology and pathology. VEGF stimulates NO production, proposed to be a result of phosphorylation of Ser-1177 on eNOS, a residue also phosphorylated upon AMPK activation in cultured endothelial cells. The present study, utilising HAEC as a model, provides the first demonstration that AMPK is activated by physiological concentrations of VEGF; and furthermore, partially mediates VEGF-stimulated phosphorylation of eNOS on Ser-1177 and subsequent NO production. In addition, the present investigation demonstrates that the upstream AMPK kinase CaMKK is responsible for these VEGF-mediated effects. VEGF is known to increase intracellular calcium levels in endothelial cells via the generation of DAG and IP3. DAG increases Ca2+ influx through a family of non-selective cation channels, whereas IP3 promotes the release of Ca2+ from intracellular stores. High potassium-induced depolarisation, which reduces the driving force for Ca2+ entry through non-selective cation channels in endothelial cells, abolished VEGF-mediated AMPK activation, whereas the IP3 receptor blocker 2-APB was without effect. Exposure of HAEC to a DAG mimetic (OAG) also stimulated AMPK, an effect which was sensitive to the CaMKK inhibitor STO-609 and high potassium induced depolarization. The functional effects of VEGF-stimulated AMPK were also assessed in HAEC. Ablation of AMPK abrogated VEGF-stimulated HAEC migration and proliferation, two key features of the angiogenic process. While AMPK was necessary for VEGF-stimulated endothelial cell proliferation direct activation of the kinase was insufficient to induce this process. AICAR-stimulated AMPK activation has been demonstrated to stimulate fatty acid oxidation in endothelial cells. However, exposure of HAEC to VEGF did not alter fatty acid oxidation in the present study. Together, the current investigation suggests that a VEGF-Ca2+-CaMKK-AMPK-eNOS- NO pathway is present in HAEC, and furthermore, that AMPK is required, albeit insufficient, for the VEGF-stimulated angiogenic response.

Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a barrier that separates these microorganisms from the intestinal epithelium 1 . Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate degradation of the complex O -glycans found in mucins. In the distal colon, these glycans are heavily sulfated, but specific sulfatases that are active on colonic mucins have not been identified. Here we show that sulfatases are essential to the utilization of distal colonic mucin O -glycans by the human gut symbiont Bacteroides thetaiotaomicron . We characterized the activity of 12 different sulfatases produced by this species, showing that they are collectively active on all known sulfate linkages in O -glycans. Crystal structures of three enzymes provide mechanistic insight into the molecular basis of substrate specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O -glycans in vitro and also has a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by a prominent group of gut bacteria, an important process for both normal microbial gut colonization 2 and diseases such as inflammatory bowel disease 3 . A single sulfatase produced by a bacterium found in the human colon is essential for degradation of sulfated O -glycans in secreted mucus.

Summary Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a physical barrier that separates these microbes from the intestinal epithelium. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate the degradation of the highly complex O-glycans found in mucins. In the colon, these glycans are heavily sulfated, but the specific sulfatases that are active on colonic mucins have not been identified. Here, we show that sulfatases are essential to the utilization of colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We have characterized the activity of 12 different sulfatases encoded by this species, showing that these enzymes collectively are active on all of the known sulfate linkages in colonic O-glycans. Crystal structures of 3 enzymes provide mechanistic insight into the molecular basis of substrate-specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also plays a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by gut bacteria, an important process for both normal microbial gut colonization and diseases such as inflammatory bowel disease (IBD). Sulfatase activity is likely to be a keystone step in bacterial mucin degradation and inhibition of these enzymes may therefore represent a viable therapeutic path for treatment of IBD and other diseases. Competing Interest Statement The authors have declared no competing interest.