Laura E. Edgington-Mitchell

Probes to Monitor Activity of the Paracaspase MALT1

The human paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) plays a central role in nuclear factor-κB (NF-κB) signaling as both a protease and scaffolding protein. Knocking out MALT1 leads to impaired NF-κB signaling and failure to mount an effective immune response. However, it is unclear to which degree it is the scaffolding function versus the proteolytic activity of MALT1 that is essential. Previous work involving a MALT1 inhibitor with low selectivity suggests that the enzymatic function plays an important role in different cell lines. To help elucidate this proteolytic role of MALT1, we have designed activity-based probes that inhibit its proteolytic activity. The probes selectively label active enzyme and can be used to inhibit MALT1 and trace its activity profile, helping to create a better picture of the significance of the proteolytic function of MALT1.

Cysteine cathepsin activity suppresses osteoclastogenesis of myeloid-derived suppressor cells in breast cancer

Cysteine cathepsin proteases contribute to many normal cellular functions, and their aberrant activity within various cell types can contribute to many diseases, including breast cancer. It is now well accepted that cathepsin proteases have numerous cell-specific functions within the tumor microenvironment that function to promote tumor growth and invasion, such that they may be valid targets for anti-metastatic therapeutic approaches. Using activity-based probes, we have examined the activity and expression of cysteine cathepsins in a mouse model of breast cancer metastasis to bone. In mice bearing highly metastatic tumors, we detected abundant cysteine cathepsin expression and activity in myeloid-derived suppressor cells (MDSCs). These immature immune cells have known metastasis-promoting roles, including immunosuppression and osteoclastogenesis, and we assessed the contribution of cysteine cathepsins to these functions. Blocking cysteine cathepsin activity with multiple small-molecule inhibitors resulted in enhanced differentiation of multinucleated osteoclasts. This highlights a potential role for cysteine cathepsin activity in suppressing the fusion of osteoclast precursor cells. In support of this hypothesis, we found that expression and activity of key cysteine cathepsins were downregulated during MDSC-osteoclast differentiation. Another cysteine protease, legumain, also inhibits osteoclastogenesis, in part through modulation of cathepsin L activity. Together, these data suggest that cysteine protease inhibition is associated with enhanced osteoclastogenesis, a process that has been implicated in bone metastasis.

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.

Antagonism of the proinflammatory and pronociceptive actions of canonical and biased agonists of protease-activated receptor-2

Diverse proteases cleave protease‐activated receptor‐2 (PAR2) on primary sensory neurons and epithelial cells to evoke pain and inflammation. Trypsin and tryptase activate PAR2 by a canonical mechanism that entails cleavage within the extracellular N‐terminus revealing a tethered ligand that activates the cleaved receptor. Cathepsin‐S and elastase are biased agonists that cleave PAR2 at different sites to activate distinct signalling pathways. Although PAR2 is a therapeutic target for inflammatory and painful diseases, the divergent mechanisms of proteolytic activation complicate the development of therapeutically useful antagonists.

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

Long noncoding RNAs: novel links to inflammatory bowel disease?

inflammatory bowel diseases (IBDs), comprising ulcerative colitis and Crohns disease, are still largely idiopathic in nature. With the global incidence of these conditions on the rise, there is a pressing need to increase our understanding of their etiology and to develop improved therapeutic

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.

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

Significance Activated G protein-coupled receptors (GPCRs) internalize and can continue to signal from endosomes. The contribution of endosomal signaling to human disease is unknown. Proteases that are generated in the colon of patients with irritable bowel syndrome (IBS) can cleave protease-activated receptor-2 (PAR2) on nociceptors to cause pain. We evaluated whether PAR2 generates signals in endosomes of nociceptors that mediate persistent hyperexcitability and pain. Biopsies of colonic mucosa from IBS patients released proteases that induced PAR2 endocytosis, endosomal signaling, and persistent hyperexcitability of nociceptors. When conjugated to the transmembrane lipid cholestanol, PAR2 antagonists accumulated in endosomes and suppressed persistent hyperexcitability. The results reveal the therapeutic potential of endosomally targeted PAR2 antagonists for IBS pain, and expand the contribution of endosomal GPCR signaling to encompass processes that are relevant to disease. 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 β-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.

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

SUMMARY 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. Graphical Abstract Figure. No caption available. HighlightsAsparaginyl endopeptidase (AEP) is expressed in induced regulatory T cellsAEP cleaves Foxp3 and Aep−/− mice have elevated numbers of peripheral Treg cellsAEP deficiency increases Treg cell frequency and numbers in GvHD and melanomaPD‐1 signaling maintains Foxp3 protein expression by inhibiting AEP &NA; Th1 cells are known for their enhanced stability, so mechanisms that mediate their flexibility are poorly studied. Here, Stathopoulou et al. demonstrate that plasticity of Th1 cells to Tbet+iTreg cells is mediated by PD‐1 signaling via asparaginyl endopeptidase (AEP). AEP inhibition enhanced iTreg cells in GvHD and tumor models.

Detection of Active Caspases During Apoptosis Using Fluorescent Activity-Based Probes

Activity-based probes (ABPs) are reactive small molecules that covalently bind to active enzymes. When tagged with a fluorophore, ABPs serve as powerful tools to investigate enzymatic activity across a wide variety of applications. In this chapter, we provide detailed methods for using fluorescent ABPs to detect the activity of caspases during the onset of apoptosis in vitro. We describe how these probes can be used to biochemically profile caspase activity in vitro using fluorescent SDS-PAGE as well as their application to imaging protease activity in live animals and tissues.