Biochemistry and Pharmacology - Research Publications

Permanent URI for this collection

Search Results

Now showing 1 - 10 of 26
  • Item
    No Preview Available
    Chemical Tools to Image the Activity of PAR-Cleaving Proteases
    Lee, IY ; Tantisirivat, P ; Edgington-Mitchell, LE (AMER CHEMICAL SOC, 2023-05-27)
    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.
  • Item
    No Preview Available
    PROTEASE-ACTIVATED RECEPTORS IN HEALTH AND DISEASE
    Peach, CJ ; Edgington-Mitchell, LE ; Bunnett, NW ; Schmidt, BL (AMER PHYSIOLOGICAL SOC, 2023-01)
    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.
  • Item
    No Preview Available
    Eat, prey, love: Pathogen-mediated subversion of lysosomal biology.
    Bird, LE ; Edgington-Mitchell, LE ; Newton, HJ (Elsevier BV, 2023-08)
    The mammalian lysosome is classically considered the 'garbage can' of the cell, contributing to clearance of infection through its primary function as a degradative organelle. Intracellular pathogens have evolved several strategies to evade contact with this harsh environment through subversion of endolysosomal trafficking or escape into the cytosol. Pathogens can also manipulate pathways that lead to lysosomal biogenesis or alter the abundance or activity of lysosomal content. This pathogen-driven subversion of lysosomal biology is highly dynamic and depends on a range of factors, including cell type, stage of infection, intracellular niche and pathogen load. The growing body of literature in this field highlights the nuanced and complex relationship between intracellular pathogens and the host lysosome, which is critical for our understanding of infection biology.
  • Item
    Thumbnail Image
    Legumain Induces Oral Cancer Pain by Biased Agonism of Protease-Activated Receptor-2
    Tu, NH ; Jensen, DD ; Anderson, BM ; Chen, E ; Jimenez-Vargas, NN ; Scheff, NN ; Inoue, K ; Tran, HD ; Dolan, JC ; Meek, TA ; Hollenberg, MD ; Liu, CZ ; Vanner, SJ ; Janal, MN ; Bunnett, NW ; Edgington-Mitchell, LE ; Schmidt, BL (SOC NEUROSCIENCE, 2021-01-06)
    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 β-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 β-arrestin recruitment or PAR2 endocytosis. Thus, Lgmn is a biased agonist of PAR2 that evokes cancer pain.
  • Item
    Thumbnail Image
    Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86
    Liu, H ; Wilson, KR ; Firth, AM ; Macri, C ; Schriek, P ; Blum, AB ; Villar, J ; Wormald, S ; Shambrook, M ; Xu, B ; Lim, HJ ; McWilliam, HEG ; Hill, AF ; Edgington-Mitchell, LE ; Caminschi, I ; Lahoud, MH ; Segura, E ; Herold, MJ ; Villadangos, JA ; Mintern, JD (NATURE PORTFOLIO, 2022-04-11)
    The MARCH E3 ubiquitin (Ub) ligase MARCH1 regulates trafficking of major histocompatibility complex class II (MHC II) and CD86, molecules of critical importance to immunity. Here we show, using a genome-wide CRISPR knockout screen, that ubiquitin-like protein 3 (UBL3) is a necessary component of ubiquitination-mediated trafficking of these molecules in mice and in humans. Ubl3-deficient mice have elevated MHC II and CD86 expression on the surface of professional and atypical antigen presenting cells. UBL3 also regulates MHC II and CD86 in human dendritic cells (DCs) and macrophages. UBL3 impacts ubiquitination of MARCH1 substrates, a mechanism that requires UBL3 plasma membrane anchoring via prenylation. Loss of UBL3 alters adaptive immunity with impaired development of thymic regulatory T cells, loss of conventional type 1 DCs, increased number of trogocytic marginal zone B cells, and defective in vivo MHC II and MHC I antigen presentation. In summary, we identify UBL3 as a conserved, critical factor in MARCH1-mediated ubiquitination with important roles in immune responses.
  • Item
    Thumbnail Image
    Hydroxychloroquine inhibits the mitochondrial antioxidant system in activated T cells
    Kim, ML ; Hardy, MY ; Edgington-Mitchell, LE ; Ramarathinam, SH ; Chung, SZ ; Russell, AK ; Currie, I ; Sleebs, BE ; Purcell, AW ; Tye-Din, JA ; Wicks, IP (CELL PRESS, 2021-12-17)
    Although hydroxychloroquine (HCQ) has long been used to treat autoimmune diseases, its mechanism of action remains poorly understood. In CD4 T-cells, we found that a clinically relevant concentration of HCQ inhibited the mitochondrial antioxidant system triggered by TCR crosslinking, leading to increased mitochondrial superoxide, impaired activation-induced autophagic flux, and reduced proliferation of CD4 T-cells. In antigen-presenting cells, HCQ also reduced constitutive activation of the endo-lysosomal protease legumain and toll-like receptor 9, thereby reducing cytokine production, but it had little apparent impact on constitutive antigen processing and peptide presentation. HCQ's effects did not require endo-lysosomal pH change, nor impaired autophagosome-lysosome fusion. We explored the clinical relevance of these findings in patients with celiac disease-a prototypic CD4 T-cell-mediated disease-and found that HCQ limits ex vivo antigen-specific T cell responses. We report a T-cell-intrinsic immunomodulatory effect from HCQ and suggest potential re-purposing of HCQ for celiac disease.
  • Item
    No Preview Available
    N-Terminomics/TAILS Profiling of Proteases and Their Substrates in Ulcerative Colitis
    Gordon, MH ; Anowai, A ; Young, D ; Das, N ; Campden, RI ; Sekhon, H ; Myers, Z ; Mainoli, B ; Chopra, S ; Thuy-Boun, PS ; Kizhakkedathu, J ; Bindra, G ; Jijon, HB ; Heitman, S ; Yates, R ; Wolan, DW ; Edgington-Mitchell, LE ; MacNaughton, WK ; Dufour, A (AMER CHEMICAL SOC, 2019-11)
    Dysregulated protease activity is often implicated in the initiation of inflammation and immune cell recruitment in gastrointestinal inflammatory diseases. Using N-terminomics/TAILS (terminal amine isotopic labeling of substrates), we compared proteases, along with their substrates and inhibitors, between colonic mucosal biopsies of healthy patients and those with ulcerative colitis (UC). Among the 1642 N-termini enriched using TAILS, increased endogenous processing of proteins was identified in UC compared to healthy patients. Changes in the reactome pathways for proteins associated with metabolism, adherens junction proteins (E-cadherin, liver-intestinal cadherin, catenin alpha-1, and catenin delta-1), and neutrophil degranulation were identified between the two groups. Increased neutrophil infiltration and distinct proteases observed in ulcerative colitis may result in extensive break down, altered processing, or increased remodeling of adherens junctions and other cellular functions. Analysis of the preferred proteolytic cleavage sites indicated that the majority of proteolytic activity and processing comes from host proteases, but that key microbial proteases may also play a role in maintaining homeostasis. Thus, the identification of distinct proteases and processing of their substrates improves the understanding of dysregulated proteolysis in normal intestinal physiology and ulcerative colitis.
  • Item
    Thumbnail Image
    Cathepsin S Evokes PAR2-Dependent Pain in Oral Squamous Cell Carcinoma Patients and Preclinical Mouse Models
    Nguyen, HT ; Inoue, K ; Chen, E ; Anderson, BM ; Sawicki, CM ; Scheff, NN ; Tran, HD ; Kim, DH ; Alemu, RG ; Yang, L ; Dolan, JC ; Liu, CZ ; Janal, MN ; Latorre, R ; Jensen, DD ; Bunnett, NW ; Edgington-Mitchell, LE ; Schmidt, BL (MDPI, 2021-09)
    Oral squamous cell carcinoma (SCC) pain is more prevalent and severe than pain generated by any other form of cancer. We previously showed that protease-activated receptor-2 (PAR2) contributes to oral SCC pain. Cathepsin S is a lysosomal cysteine protease released during injury and disease that can activate PAR2. We report here a role for cathepsin S in PAR2-dependent cancer pain. We report that cathepsin S was more active in human oral SCC than matched normal tissue, and in an orthotopic xenograft tongue cancer model than normal tongue. The multiplex immunolocalization of cathepsin S in human oral cancers suggests that carcinoma and macrophages generate cathepsin S in the oral cancer microenvironment. After cheek or paw injection, cathepsin S evoked nociception in wild-type mice but not in mice lacking PAR2 in Nav1.8-positive neurons (Par2Nav1.8), nor in mice treated with LY3000328 or an endogenous cathepsin S inhibitor (cystatin C). The human oral SCC cell line (HSC-3) with homozygous deletion of the gene for cathepsin S (CTSS) with CRISPR/Cas9 provoked significantly less mechanical allodynia and thermal hyperalgesia, as did those treated with LY3000328, compared to the control cancer mice. Our results indicate that cathepsin S is activated in oral SCC, and that cathepsin S contributes to cancer pain through PAR2 on neurons.
  • Item
    Thumbnail Image
    Antagonism of the proinflammatory and pronociceptive actions of canonical and biased agonists of protease-activated receptor-2
    Lieu, T ; Savage, E ; Zhao, P ; Edgington-Mitchell, L ; Barlow, N ; Bron, R ; Poole, DP ; McLean, P ; Lohman, R-J ; Fairlie, DP ; Bunnett, NW (WILEY, 2016-09)
    BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: We investigated whether the PAR2 antagonist GB88 inhibits protease-evoked activation of nociceptors and protease-stimulated oedema and hyperalgesia in rodents. KEY RESULTS: Intraplantar injection of trypsin, cathespsin-S or elastase stimulated mechanical and thermal hyperalgesia and oedema in mice. Oral GB88 or par2 deletion inhibited the algesic and proinflammatory actions of all three proteases, but did not affect basal responses. GB88 also prevented pronociceptive and proinflammatory effects of the PAR2-selective agonists 2-furoyl-LIGRLO-NH2 and AC264613. GB88 did not affect capsaicin-evoked hyperalgesia or inflammation. Trypsin, cathepsin-S and elastase increased [Ca(2+) ]i in rat nociceptors, which expressed PAR2. GB88 inhibited this activation of nociceptors by all three proteases, but did not affect capsaicin-evoked activation of nociceptors or inhibit the catalytic activity of the three proteases. CONCLUSIONS AND IMPLICATIONS: GB88 inhibits the capacity of canonical and biased protease agonists of PAR2 to cause nociception and inflammation.
  • Item
    Thumbnail Image
    Cysteine cathepsin activity suppresses osteoclastogenesis of myeloid-derived suppressor cells in breast cancer
    Edgington-Mitchell, LE ; Rautela, J ; Duivenvoorden, HM ; Jayatilleke, KM ; van der Linden, WA ; Verdoes, M ; Bogyo, M ; Parker, BS (IMPACT JOURNALS LLC, 2015-09-29)
    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.