54 results on '"Laura E. Edgington-Mitchell"'
Search Results
2. Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86
- Author
-
Haiyin Liu, Kayla R. Wilson, Ashley M. Firth, Christophe Macri, Patrick Schriek, Annabelle B. Blum, Javiera Villar, Samuel Wormald, Mitch Shambrook, Bangyan Xu, Hui Jing Lim, Hamish E. G. McWilliam, Andrew F. Hill, Laura E. Edgington-Mitchell, Irina Caminschi, Mireille H. Lahoud, Elodie Segura, Marco J. Herold, Jose A. Villadangos, and Justine D. Mintern more...
- Subjects
Science - Abstract
Regulated trafficking of major histocompatibility complex class II and CD86 is a prerequisite of antigen presenting cell functionality. Authors show here that ubiquitin-like protein 3 is critically involved in the ubiquitination process that controls trafficking, with wide-ranging immunological consequences. more...
- Published
- 2022
- Full Text
- View/download PDF
Catalog
3. High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections – Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils
- Author
-
Niina Aaltonen, Prosanta K. Singha, Hermina Jakupović, Thomas Wirth, Haritha Samaranayake, Sanna Pasonen-Seppänen, Kirsi Rilla, Markku Varjosalo, Laura E. Edgington-Mitchell, Paulina Kasperkiewicz, Marcin Drag, Sara Kälvälä, Eemeli Moisio, Juha R. Savinainen, and Jarmo T. Laitinen more...
- Subjects
Activity-based protein profiling (ABPP) ,Brain cryosection ,Glioblastoma multiforme (GBM) ,Immunohistochemistry ,Serine hydrolase activity ,Neutrophil serine protease (NSP) ,Medicine (General) ,R5-920 ,Biology (General) ,QH301-705.5 - Abstract
Abstract 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. more...
- Published
- 2020
- Full Text
- View/download PDF
4. Hydroxychloroquine inhibits the mitochondrial antioxidant system in activated T cells
- Author
-
Man Lyang Kim, Melinda Y. Hardy, Laura E. Edgington-Mitchell, Sri H. Ramarathinam, Shan Zou Chung, Amy K. Russell, Iain Currie, Brad E. Sleebs, Anthony W. Purcell, Jason A. Tye-Din, and Ian P. Wicks
- Subjects
Molecular biology ,Immune system ,Proteomics ,Science - Abstract
Summary: 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. more...
- Published
- 2021
- Full Text
- View/download PDF
5. Loss of O-Linked Protein Glycosylation in Burkholderia cenocepacia Impairs Biofilm Formation and Siderophore Activity and Alters Transcriptional Regulators
- Author
-
Cameron C. Oppy, Leila Jebeli, Miku Kuba, Clare V. Oates, Richard Strugnell, Laura E. Edgington-Mitchell, Miguel A. Valvano, Elizabeth L. Hartland, Hayley J. Newton, and Nichollas E. Scott
- Subjects
glycosylation ,pathogenesis ,Burkholderia cenocepacia ,posttranslational modifications ,proteomics ,DNA binding ,Microbiology ,QR1-502 - Abstract
ABSTRACT O-linked protein glycosylation is a conserved feature of the Burkholderia genus. The addition of the trisaccharide β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc to membrane exported proteins in Burkholderia cenocepacia is required for bacterial fitness and resistance to environmental stress. However, the underlying causes of the defects observed in the absence of glycosylation are unclear. Using proteomics, luciferase reporter assays, and DNA cross-linking, we demonstrate the loss of glycosylation leads to changes in transcriptional regulation of multiple proteins, including the repression of the master quorum CepR/I. These proteomic and transcriptional alterations lead to the abolition of biofilm formation and defects in siderophore activity. Surprisingly, the abundance of most of the known glycosylated proteins did not significantly change in the glycosylation-defective mutants, except for BCAL1086 and BCAL2974, which were found in reduced amounts, suggesting they could be degraded. However, the loss of these two proteins was not responsible for driving the proteomic alterations, biofilm formation, or siderophore activity. Together, our results show that loss of glycosylation in B. cenocepacia results in a global cell reprogramming via alteration of the transcriptional regulatory systems, which cannot be explained by the abundance changes in known B. cenocepacia glycoproteins. IMPORTANCE Protein glycosylation is increasingly recognized as a common posttranslational protein modification in bacterial species. Despite this commonality, our understanding of the role of most glycosylation systems in bacterial physiology and pathogenesis is incomplete. In this work, we investigated the effect of the disruption of O-linked glycosylation in the opportunistic pathogen Burkholderia cenocepacia using a combination of proteomic, molecular, and phenotypic assays. We find that in contrast to recent findings on the N-linked glycosylation systems of Campylobacter jejuni, O-linked glycosylation does not appear to play a role in proteome stabilization of most glycoproteins. Our results reveal that loss of glycosylation in B. cenocepacia strains leads to global proteome and transcriptional changes, including the repression of the quorum-sensing regulator cepR (BCAM1868) gene. These alterations lead to dramatic phenotypic changes in glycosylation-null strains, which are paralleled by both global proteomic and transcriptional alterations, which do not appear to directly result from the loss of glycosylation per se. This research unravels the pleiotropic effects of O-linked glycosylation in B. cenocepacia, demonstrating that its loss does not simply affect the stability of the glycoproteome, but also interferes with transcription and the broader proteome. more...
- Published
- 2019
- Full Text
- View/download PDF
6. Sez6 levels are elevated in cerebrospinal fluid of patients with inflammatory pain–associated conditions
- Author
-
Maria Roitman, Laura E. Edgington-Mitchell, Jon Mangum, James Ziogas, Alexios A. Adamides, Paul Myles, Hearan Choo-Bunnett, Nigel W. Bunnett, and Jenny M. Gunnersen
- Subjects
Anesthesiology ,RD78.3-87.3 - Abstract
Abstract. Introduction:. Seizure-related protein 6 (Sez6) contributes to chronic pain development as sez6 knockout mice show attenuated pain behaviours after peripheral nerve injury, compared with control mice. The type I transmembrane isoform of Sez6 is cleaved by the β-amyloid precursor protein cleavage enzyme 1 (BACE1), resulting in Sez6 extracellular domain shedding from the neuron surface. Objectives:. To determine whether this BACE1-shed form of Sez6 can be detected in the cerebrospinal fluid (CSF) and whether Sez6 levels in the CSF are altered in neuropathic pain or chronic inflammatory pain (IP). Methods:. We analysed the CSF samples collected during surgery from patients with chronic neuropathic pain (n = 8) or IP (n = 33), comparing them to the CSF samples from patients with suspected subarachnoid haemorrhage that was subsequently excluded (nonsurgical group, n = 5). Western blots were used to determine the relative Sez6 levels in the CSF from the different patient and nonsurgical comparison groups. Results:. The results show that BACE1-shed Sez6 can be readily detected in the CSF by Western blot and that the levels of Sez6 are significantly higher in the IP group than in the nonsurgical comparison group. Conclusion:. The association between elevated Sez6 levels in the CSF and IP is further evidence for persistent alterations in central nervous system activity in chronic IP conditions. more...
- Published
- 2019
- Full Text
- View/download PDF
7. Protease-activated receptors in health and disease
- Author
-
Chloe J. Peach, Laura E. Edgington-Mitchell, Nigel W. Bunnett, and Brian L. Schmidt
- Subjects
Physiology ,Physiology (medical) ,Receptors, Proteinase-Activated ,Humans ,Homeostasis ,General Medicine ,Molecular Biology ,Signal Transduction ,Receptors, G-Protein-Coupled ,Peptide Hydrolases - Abstract
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. more...
- Published
- 2024
8. Chemical Tools to Image the Activity of PAR-Cleaving Proteases
- Author
-
Irene Y. Lee, Piyapa Tantisirivat, and Laura E. Edgington-Mitchell
- Subjects
Drug Discovery ,Pharmaceutical Science ,Molecular Biology ,Biochemistry - Published
- 2023
- Full Text
- View/download PDF
9. Supplementary figs from C/EBPβ/AEP Signaling Regulates the Oxidative Stress in Malignant Cancers, Stimulating the Metastasis
- Author
-
Keqiang Ye, Lingjing Jin, Laura E. Edgington-Mitchell, Hua Li, Xia Liu, Jianming Liao, Chun Chen, Eun Hee Ahn, Seong Su Kang, and Kecheng Lei
- Abstract
Supplementary figs
- Published
- 2023
- Full Text
- View/download PDF
10. Data from C/EBPβ/AEP Signaling Regulates the Oxidative Stress in Malignant Cancers, Stimulating the Metastasis
- Author
-
Keqiang Ye, Lingjing Jin, Laura E. Edgington-Mitchell, Hua Li, Xia Liu, Jianming Liao, Chun Chen, Eun Hee Ahn, Seong Su Kang, and Kecheng Lei
- Abstract
Solid tumors start as a local disease, but some are capable of metastasizing to the lymph nodes and distant organs. The hypoxic microenvironment, which is critical during cancer development, plays a key role in regulating cancer progression and metastasis. However, the molecular mechanisms mediating the disseminated cancer cell metastasis remain incompletely understood. Here, we show that C/EBPβ/AEP signaling that is upregulated in breast cancers mediates oxidative stress and lung metastasis, and inactivation of asparagine endopeptidase (AEP, also known as legumain) robustly regulates breast cancer reactive oxygen species (ROS) and metastasis. AEP, a protease activated in acidic conditions, is overexpressed in numerous types of cancer and promotes metastasis. Employing a breast cancer cell line MDA-MD-231, we show that C/EBPβ, an oxidative stress or inflammation-activated transcription factor, and its downstream target AEP mediate ROS production as well as migration and invasion in cancer cells. Deficiency of AEP in the MMTV-PyMT transgenic breast cancer mouse model significantly regulates oxidative stress and suppresses lung metastasis. Administration of an innovative AEP inhibitor substantially mitigates ROS production and cancer metastasis. Hence, our study demonstrates that pharmacologic inhibition of AEP activity might provide a disease-modifying strategy to suppress cancer metastasis. more...
- Published
- 2023
- Full Text
- View/download PDF
11. MHC Class II Ubiquitination Regulates Dendritic Cell Function and Immunity
- Author
-
F Victor Makota, Haiyin Liu, Jose A Villadangos, Kayla R. Wilson, Satoshi Ishido, Mireille H. Lahoud, Annabelle B. Blum, Christophe Macri, Scott N. Mueller, Justine D. Mintern, Devi Jenika, Patrick Schriek, Bangyan Xu, Irina Caminschi, Dominik Schienstock, Laura E Edgington-Mitchell, and Lauren Francis more...
- Subjects
CD4-Positive T-Lymphocytes ,Cytotoxicity, Immunologic ,medicine.medical_treatment ,T cell ,Immunology ,Mice, Transgenic ,chemical and pharmacologic phenomena ,Lymphocyte Activation ,Major histocompatibility complex ,Mice ,MHC class I ,medicine ,Animals ,Immunology and Allergy ,Receptor ,Cells, Cultured ,Antigen Presentation ,Immunity, Cellular ,MHC class II ,biology ,Chemistry ,Histocompatibility Antigens Class II ,Ubiquitination ,Dendritic Cells ,Dendritic cell ,Germinal Center ,Molecular biology ,Mice, Inbred C57BL ,CTL ,Cytokine ,medicine.anatomical_structure ,Mutation ,biology.protein - Abstract
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. more...
- Published
- 2021
- Full Text
- View/download PDF
12. Eat, prey, love: Pathogen-mediated subversion of lysosomal biology
- Author
-
Lauren E Bird, Laura E Edgington-Mitchell, and Hayley J Newton
- Subjects
Immunology ,Immunology and Allergy - Published
- 2023
- Full Text
- View/download PDF
13. Legumain Induces Oral Cancer Pain by Biased Agonism of Protease-Activated Receptor-2
- Author
-
Morley D. Hollenberg, Cheng Z. Liu, Elyssa Chen, Stephen Vanner, Dane D. Jensen, Nicole N. Scheff, Malvin N. Janal, Nigel W. Bunnett, Kenji Inoue, Hung D. Tran, Bethany M. Anderson, Nestor N. Jiménez-Vargas, Tamaryn A. Meek, Laura E. Edgington-Mitchell, Brian L. Schmidt, Nguyen Huu Tu, and John C. Dolan more...
- Subjects
Male ,0301 basic medicine ,Legumain ,Endocytosis ,Adenylyl cyclase ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Tumor Microenvironment ,medicine ,Animals ,Humans ,Receptor, PAR-2 ,Protein kinase A ,Protein Kinase Inhibitors ,Research Articles ,Protein Kinase C ,Protease-activated receptor 2 ,Aged ,Aged, 80 and over ,Mice, Knockout ,Arrestin ,biology ,Chemistry ,Beta-Arrestins ,General Neuroscience ,Cancer ,Cancer Pain ,Middle Aged ,medicine.disease ,Cyclic AMP-Dependent Protein Kinases ,Enzyme Activation ,Mice, Inbred C57BL ,Cysteine Endopeptidases ,stomatognathic diseases ,030104 developmental biology ,Cell culture ,Carcinoma, Squamous Cell ,biology.protein ,Cancer research ,Female ,Mouth Neoplasms ,030217 neurology & neurosurgery - Abstract
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 PAR2in 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.Par2Nav1.8andLgmndeletion attenuated mechanical allodynia in female mice with carcinogen-induced OSCC. Lgmn caused PAR2-dependent hyperexcitability of trigeminal neurons from WT female mice.Par2deletion, 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 PAR2at Asn30↓Arg31, proximal to the canonical trypsin activation site. Lgmn activated PAR2by biased mechanisms in HEK293 cells to induce Ca2+mobilization, cAMP formation, and PKA/protein kinase D (PKD) activation, but not β-arrestin recruitment or PAR2endocytosis. Thus, in the acidified OSCC microenvironment, Lgmn activates PAR2by biased mechanisms that evoke cancer pain.SIGNIFICANCE STATEMENTOral 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 PAR2to induce calcium mobilization, cAMP formation, and activation of protein kinase D (PKD) and PKA, but not β-arrestin recruitment or PAR2endocytosis. Thus, Lgmn is a biased agonist of PAR2that evokes cancer pain. more...
- Published
- 2020
- Full Text
- View/download PDF
14. TrkB receptor cleavage by delta-secretase abolishes its phosphorylation of APP, aggravating Alzheimer’s disease pathologies
- Author
-
Keqiang Ye, Yiyuan Xia, Pai Liu, Xia Liu, Xiaochuan Wang, Laura E. Edgington-Mitchell, and Zhi-Hao Wang
- Subjects
0301 basic medicine ,biology ,Kinase ,Chemistry ,musculoskeletal, neural, and ocular physiology ,Tropomyosin receptor kinase B ,Cell biology ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Psychiatry and Mental health ,030104 developmental biology ,0302 clinical medicine ,nervous system ,Neurotrophic factors ,mental disorders ,embryonic structures ,Synaptic plasticity ,Amyloid precursor protein ,biology.protein ,Phosphorylation ,Molecular Biology ,Amyloid precursor protein secretase ,030217 neurology & neurosurgery ,Neurotrophin - Abstract
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-β 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. more...
- Published
- 2020
- Full Text
- View/download PDF
15. Lysosomal degradation products induce Coxiella burnetii virulence
- Author
-
Kaylene J. Simpson, Patrice Newton, Hayley J. Newton, Piyush B. Madhamshettiwar, Shawna C. O. Reed, Nicole Lau, Laura E. Edgington-Mitchell, Craig R. Roy, Sze Ying Ong, Bangyan Xu, Shivani Pasricha, and David R. Thomas more...
- Subjects
0303 health sciences ,Reporter gene ,Multidisciplinary ,030306 microbiology ,Effector ,Virulence ,Vacuole ,Biology ,bacterial infections and mycoses ,Coxiella burnetii ,biology.organism_classification ,Cell biology ,03 medical and health sciences ,medicine.anatomical_structure ,Regulon ,Lysosome ,medicine ,bacteria ,Secretion ,030304 developmental biology - Abstract
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. more...
- Published
- 2020
- Full Text
- View/download PDF
16. Cathepsin S Evokes PAR
- Author
-
Nguyen Huu, Tu, Kenji, Inoue, Elyssa, Chen, Bethany M, Anderson, Caroline M, Sawicki, Nicole N, Scheff, Hung D, Tran, Dong H, Kim, Robel G, Alemu, Lei, Yang, John C, Dolan, Cheng Z, Liu, Malvin N, Janal, Rocco, Latorre, Dane D, Jensen, Nigel W, Bunnett, Laura E, Edgington-Mitchell, and Brian L, Schmidt more...
- Subjects
oral squamous cell carcinoma ,stomatognathic diseases ,cancer pain ,cathepsin S ,protease-activated receptor-2 ,pain ,oral cancer ,PAR2 ,Article - Abstract
Simple Summary Oral cancer is often deadly and severely painful. Because this form of cancer pain cannot be adequately treated with current medications including opiates, new treatment approaches are needed. Cathepsin S, a lysosomal cysteine protease may play a role in oral cancer pain through a protease-activated receptor-2 (PAR2)-dependent mechanism. We undertook a series of experiments to define the role of cathepsin S in oral cancer pain. We compared cathepsin S activity in human oral cancer tumors versus patient-matched normal tissue; a human oral cancer cell line versus a benign dysplastic oral keratinocyte cell line; and in an orthotopic xenograft tongue cancer mouse model versus normal controls in mice. We localized cathepsin S in macrophages and carcinoma cells in human oral cancers. The injection of cathepsin S caused nociception in a mouse model while the injection of oral cancer cells in which the gene for cathepsin S is deleted generated less nociception. Our findings will lay the foundations for clinical trials of cathepsin S inhibitors for treating oral cancer pain. Abstract 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. more...
- Published
- 2021
17. Hydroxychloroquine inhibits the mitochondrial antioxidant system in activated T cells
- Author
-
Shan Zou Chung, Jason A. Tye-Din, Ian P. Wicks, Iain Currie, Sri H. Ramarathinam, Man Lyang Kim, Anthony W. Purcell, Amy K. Russell, Brad E. Sleebs, Laura E. Edgington-Mitchell, and Melinda Y Hardy
- Subjects
Proteomics ,Multidisciplinary ,biology ,Molecular biology ,Chemistry ,Antigen processing ,Science ,medicine.medical_treatment ,T cell ,T-cell receptor ,Hydroxychloroquine ,Pharmacology ,Legumain ,Article ,Immune system ,Cytokine ,medicine.anatomical_structure ,biology.protein ,medicine ,Antigen-presenting cell ,Ex vivo ,medicine.drug - Abstract
Summary 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. more...
- Published
- 2021
18. N-Terminomics/TAILS Profiling of Macrophages after Chemical Inhibition of Legumain
- Author
-
Luiz Gustavo de Almeida, Antoine Dufour, Henna Sekhon, Laura E. Edgington-Mitchell, Daniel Young, and Bethany M. Anderson
- Subjects
Proteomics ,Iron ,medicine.medical_treatment ,Mitochondrion ,Legumain ,Biochemistry ,Mice ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Animals ,Humans ,Asparagine ,Shotgun proteomics ,030304 developmental biology ,0303 health sciences ,Protease ,biology ,Chemistry ,Macrophages ,Endopeptidase ,Mitochondria ,3. Good health ,Cell biology ,Cysteine Endopeptidases ,RAW 264.7 Cells ,Gene Expression Regulation ,Isotope Labeling ,030220 oncology & carcinogenesis ,biology.protein ,Frataxin ,Peptides - Abstract
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. more...
- Published
- 2019
- Full Text
- View/download PDF
19. N-Terminomics/TAILS Profiling of Proteases and Their Substrates in Ulcerative Colitis
- Author
-
Sameeksha Chopra, Gurmeet K. Bindra, Jayachandran N. Kizhakkedathu, Henna Sekhon, Laura E. Edgington-Mitchell, Steven J. Heitman, Peter S. Thuy-Boun, Anthonia Anowai, Humberto Jijon, Robin M. Yates, Daniel Young, Dennis W. Wolan, Rhiannon I. Campden, Zoe Myers, B Mainoli, Nabangshu Das, Antoine Dufour, Wallace K. MacNaughton, and Marilyn Gordon more...
- Subjects
Adult ,Male ,Proteomics ,0301 basic medicine ,Proteases ,Colon ,Biopsy ,Proteolysis ,medicine.medical_treatment ,01 natural sciences ,Biochemistry ,Mass Spectrometry ,Adherens junction ,03 medical and health sciences ,medicine ,Humans ,Protease Inhibitors ,Amino Acid Sequence ,Colitis ,Chromatography, High Pressure Liquid ,Aged ,Binding Sites ,Protease ,medicine.diagnostic_test ,010405 organic chemistry ,Cadherin ,Chemistry ,Catenins ,General Medicine ,Middle Aged ,Cadherins ,medicine.disease ,0104 chemical sciences ,3. Good health ,Cell biology ,030104 developmental biology ,Isotope Labeling ,Catenin ,Neutrophil degranulation ,Molecular Medicine ,Colitis, Ulcerative ,Female ,Peptides ,Peptide Hydrolases ,Protein Binding ,Signal Transduction - Abstract
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. more...
- Published
- 2019
- Full Text
- View/download PDF
20. Mitochondrial dysfunction triggers the pathogenesis of Parkinson's disease in neuronal C/EBPβ transgenic mice
- Author
-
Wei Hong, Lingjing Jin, Seong Su Kang, Eun Hee Ahn, Xia Liu, Keqiang Ye, Laura E. Edgington-Mitchell, Yu Tian Wang, Kecheng Lei, and Zhi-Hao Wang
- Subjects
chemistry.chemical_classification ,Reactive oxygen species ,Parkinson's disease ,Dopaminergic Neurons ,Substantia nigra ,Mice, Transgenic ,Parkinson Disease ,Oxidative phosphorylation ,medicine.disease ,Cell biology ,Mitochondria ,Pathogenesis ,Substantia Nigra ,Cellular and Molecular Neuroscience ,Psychiatry and Mental health ,Mice ,Oxidative Stress ,Mitochondrial respiratory chain ,chemistry ,Coenzyme Q – cytochrome c reductase ,medicine ,Animals ,SDHD ,Molecular Biology - Abstract
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/EBPβ 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/EBPβ/AEP pathway that triggers dopaminergic neuronal cell death. Oral administration of these inhibitors to Thy1-C/EBPβ 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/EBPβ mice triggers ROS and PD pathologies, resulting in motor disorders. Hence, our findings demonstrate that mitochondrial ETC inactivation triggers PD pathogenesis via activating C/EBPβ/AEP pathway. more...
- Published
- 2021
21. Editorial overview: Systems biology and the rise and rise of omics approaches
- Author
-
Laura E. Edgington-Mitchell and Nichollas E. Scott
- Subjects
Engineering ,business.industry ,Systems biology ,Systems Biology ,MEDLINE ,Computational Biology ,Genomics ,Omics ,Biochemistry ,Data science ,Analytical Chemistry ,Humans ,Metabolomics ,business - Published
- 2021
22. C/EBPβ/AEP Signaling Regulates the Oxidative Stress in Malignant Cancers, Stimulating the Metastasis
- Author
-
Seong Su Kang, Jianming Liao, Eun Hee Ahn, Keqiang Ye, Hua Li, Chun Chen, Kecheng Lei, Laura E. Edgington-Mitchell, Lingjing Jin, and Xia Liu
- Subjects
Cancer Research ,Lung Neoplasms ,Cell ,Apoptosis ,Breast Neoplasms ,medicine.disease_cause ,Metastasis ,Mice ,Breast cancer ,Growth factor receptor ,Biomarkers, Tumor ,Tumor Cells, Cultured ,Tumor Microenvironment ,Medicine ,Animals ,Humans ,Lung cancer ,Cell Proliferation ,business.industry ,CCAAT-Enhancer-Binding Protein-beta ,Cancer ,medicine.disease ,Xenograft Model Antitumor Assays ,Gene Expression Regulation, Neoplastic ,Cysteine Endopeptidases ,Oxidative Stress ,medicine.anatomical_structure ,Oncology ,Cancer cell ,Cancer research ,Female ,business ,Reactive Oxygen Species ,Oxidative stress - Abstract
Solid tumors start as a local disease, but some are capable of metastasizing to the lymph nodes and distant organs. The hypoxic microenvironment, which is critical during cancer development, plays a key role in regulating cancer progression and metastasis. However, the molecular mechanisms mediating the disseminated cancer cell metastasis remain incompletely understood. Here, we show that C/EBPβ/AEP signaling that is upregulated in breast cancers mediates oxidative stress and lung metastasis, and inactivation of asparagine endopeptidase (AEP, also known as legumain) robustly regulates breast cancer reactive oxygen species (ROS) and metastasis. AEP, a protease activated in acidic conditions, is overexpressed in numerous types of cancer and promotes metastasis. Employing a breast cancer cell line MDA-MD-231, we show that C/EBPβ, an oxidative stress or inflammation-activated transcription factor, and its downstream target AEP mediate ROS production as well as migration and invasion in cancer cells. Deficiency of AEP in the MMTV-PyMT transgenic breast cancer mouse model significantly regulates oxidative stress and suppresses lung metastasis. Administration of an innovative AEP inhibitor substantially mitigates ROS production and cancer metastasis. Hence, our study demonstrates that pharmacologic inhibition of AEP activity might provide a disease-modifying strategy to suppress cancer metastasis. more...
- Published
- 2021
23. High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections - Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils
- Author
-
Thomas Wirth, Eemeli Moisio, Kirsi Rilla, Paulina Kasperkiewicz, Prosanta K. Singha, Markku Varjosalo, Sanna Pasonen-Seppänen, Jarmo T. Laitinen, Sara Kälvälä, Marcin Drag, Juha R. Savinainen, Hermina Jakupović, Laura E. Edgington-Mitchell, Niina Aaltonen, Haritha Samaranayake, University Management, Institute of Biotechnology, Helsinki Institute of Life Science HiLIFE, Joint Activities, and Molecular Systems Biology more...
- Subjects
0301 basic medicine ,Streptavidin ,Proteases ,Fluorescence-lifetime imaging microscopy ,Confocal ,Serine hydrolase activity ,METABOLISM ,Proteomics ,General Biochemistry, Genetics and Molecular Biology ,PROBES ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Activity-based protein profiling (ABPP) ,Brain cryosection ,lcsh:QH301-705.5 ,Tumor-associated neutrophils ,lcsh:R5-920 ,Tumor microenvironment ,Glioblastoma multiforme (GBM) ,LANDSCAPE ,Research ,Neutrophil serine protease (NSP) ,Immunohistochemistry ,Cell biology ,PROTEASES ,ABHD6 ,030104 developmental biology ,lcsh:Biology (General) ,chemistry ,030220 oncology & carcinogenesis ,DISCOVERY ,AUTORADIOGRAPHY ,Proteome ,CELLS ,TAMRA-FP probe ,PROTEOMICS ,1182 Biochemistry, cell and molecular biology ,lcsh:Medicine (General) ,INHIBITORS - Abstract
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. more...
- Published
- 2020
24. Lysosomal degradation products induce
- Author
-
Patrice, Newton, David R, Thomas, Shawna C O, Reed, Nicole, Lau, Bangyan, Xu, Sze Ying, Ong, Shivani, Pasricha, Piyush B, Madhamshettiwar, Laura E, Edgington-Mitchell, Kaylene J, Simpson, Craig R, Roy, and Hayley J, Newton more...
- Subjects
siRNA screen ,Tripeptidyl-Peptidase 1 ,Virulence ,Gene Expression Regulation, Bacterial ,Biological Sciences ,bacterial infections and mycoses ,Microbiology ,Dot/Icm secretion system ,Protein Transport ,Bacterial Proteins ,Coxiella burnetii ,amino acid sensing ,Host-Pathogen Interactions ,bacteria ,Humans ,Lysosomes ,Q Fever ,virulence regulation ,Bacterial Secretion Systems ,HeLa Cells - Abstract
Significance Coxiella burnetii is a unique bacterial pathogen that replicates to high numbers in a lysosome-like intracellular niche. This study identified host proteins that contribute to the pathogen’s capacity to establish this niche and activate the Dot/Icm secretion system required for intracellular replication. Many host proteins were found to contribute to the establishment of C. burnetii virulence by aiding trafficking of the pathogen to the lysosome and creating the degradative lysosome environment. Pathogenic bacteria are able to sense and adapt to their environment by altering their gene expression profile. Here we demonstrated that C. burnetii detects specific amino acids present in the lysosome using a two-component system that up-regulates expression of genes required for Dot/Icm activity., 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. more...
- Published
- 2020
25. Application of a Sulfoxonium Ylide Electrophile to Generate Cathepsin X-Selective Activity-Based Probes
- Author
-
Simon J. Mountford, Jiayin Diao, Bangyan Xu, Bethany M. Anderson, Erik Lindström, Philip E. Thompson, Elean S.V. Tay, Luigi Aurelio, Erica K. Sloan, Robin M. Yates, Monika Szabo, Rhiannon I. Campden, Nigel W. Bunnett, Laura E. Edgington-Mitchell, and My Linh Hoang more...
- Subjects
0301 basic medicine ,Male ,Hydrocarbons, Fluorinated ,Cell Culture Techniques ,Kidney ,01 natural sciences ,Biochemistry ,Article ,Cell Line ,Substrate Specificity ,Carboxypeptidase activity ,Small Molecule Libraries ,03 medical and health sciences ,Mice ,Structure-Activity Relationship ,Protein Domains ,In vivo ,Structure–activity relationship ,Animals ,Humans ,Amino Acids ,Enzyme Inhibitors ,Fluorescent Dyes ,Cathepsin ,chemistry.chemical_classification ,010405 organic chemistry ,Chemistry ,Optical Imaging ,General Medicine ,Ketones ,Cathepsins ,0104 chemical sciences ,3. Good health ,Amino acid ,Mice, Inbred C57BL ,Kinetics ,030104 developmental biology ,Diazomethane ,Cell culture ,Molecular Medicine ,Function (biology) ,Cysteine - Abstract
Cathepsin X/Z/P is cysteine cathepsin with unique carboxypeptidase activity. Its expression is associated with cancer and neurodegenerative diseases, although its roles during normal physiology are still poorly understood. Advances in our understanding of its function have been hindered by a lack of available tools that can specifically measure the proteolytic activity of cathepsin X. We present a series of activity-based probes that incorporate a sulfoxonium ylide warhead, which exhibit improved specificity for cathepsin X compared to previously reported probes. We apply these probes to detect cathepsin X activity in cell and tissue lysates, in live cells and in vivo, and to localize active cathepsin X in mouse tissues by microscopy. Finally, we utilize an improved method to generate chloromethylketones, necessary intermediates for synthesis of acyloxymethylketones probes, by way of sulfoxonium ylide intermediates. In conclusion, the probes presented in this study will be valuable for investigating cathepsin X pathophysiology. more...
- Published
- 2020
26. Novel broad-spectrum activity-based probes to profile malarial cysteine proteases
- Author
-
Mateo I. Sánchez, Edgar Deu, Dara Davison, Ambrosius P. Snijders, Bethany M. Anderson, Michele S. Y. Tan, Laura E. Edgington-Mitchell, and Stephen Howell
- Subjects
0301 basic medicine ,Plasmodium ,Cell Membrane Permeability ,Cell ,Biochemistry ,Broad spectrum ,0302 clinical medicine ,Cysteine Proteases ,Medicine and Health Sciences ,Sulfones ,Amino Acids ,media_common ,Protozoans ,Chemical Biology & High Throughput ,Multidisciplinary ,biology ,Chemistry ,Organic Compounds ,Tryptophan ,Malarial Parasites ,Eukaryota ,Proteases ,3. Good health ,Enzymes ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Physical Sciences ,Medicine ,Research Article ,Drug ,Model organisms ,media_common.quotation_subject ,Science ,Plasmodium falciparum ,Infectious Disease ,Cysteine Proteinase Inhibitors ,Biochemistry & Proteomics ,Antimalarials ,03 medical and health sciences ,Parasite Groups ,medicine ,Parasitic Diseases ,Animals ,Humans ,Sulfur Containing Amino Acids ,Cysteine ,Computational & Systems Biology ,Cathepsin ,Merozoites ,Organic Chemistry ,Organisms ,Chemical Compounds ,Correction ,Biology and Life Sciences ,Proteins ,Cell Biology ,biology.organism_classification ,Tropical Diseases ,Parasitic Protozoans ,Malaria ,Protein profiling ,030104 developmental biology ,Molecular Probes ,Enzymology ,Parasitology ,Apicomplexa - Abstract
Clan CA cysteine proteases, also known as papain-like proteases, play important roles throughout the malaria parasite life cycle and are therefore potential drug targets to treat this disease and prevent its transmission. In order to study the biological function of these proteases and to chemically validate some of them as viable drug targets, highly specific inhibitors need to be developed. This is especially challenging given the large number of clan CA proteases present in Plasmodium species (ten in Plasmodium falciparum), and the difficulty of designing selective inhibitors that do not cross-react with other members of the same family. Additionally, any efforts to develop antimalarial drugs targeting these proteases will also have to take into account potential off-target effects against the 11 human cysteine cathepsins. Activity-based protein profiling has been a very useful tool to determine the specificity of inhibitors against all members of an enzyme family. However, current clan CA proteases broad-spectrum activity-based probes either target endopeptidases or dipeptidyl aminopeptidases, but not both subfamilies efficiently. In this study, we present a new series of dipeptydic vinyl sulfone probes containing a free N-terminal tryptophan and a fluorophore at the P1 position that are able to label both subfamilies efficiently, both in Plasmodium falciparum and in mammalian cells, thus making them better broad-spectrum activity-based probes. We also show that some of these probes are cell permeable and can therefore be used to determine the specificity of inhibitors in living cells. Interestingly, we show that the choice of fluorophore greatly influences the specificity of the probes as well as their cell permeability. more...
- Published
- 2020
- Full Text
- View/download PDF
27. Targeting both BDNF/TrkB pathway and delta-secretase for treating Alzheimer's disease
- Author
-
Shuping Ming, Eun Hee Ahn, Chun Chen, Jianming Liao, Hua Li, Laura E. Edgington-Mitchell, Zhonghua Lu, Keqiang Ye, and Xia Liu
- Subjects
Agonist ,medicine.drug_class ,tau Proteins ,Tropomyosin receptor kinase B ,Pharmacology ,Legumain ,Neuroprotection ,Article ,Pathogenesis ,Amyloid beta-Protein Precursor ,Mice ,Cellular and Molecular Neuroscience ,Cognition ,Alzheimer Disease ,medicine ,Animals ,Humans ,Receptor, trkB ,Enzyme Inhibitors ,Maze Learning ,Protein kinase B ,Membrane Glycoproteins ,biology ,Chemistry ,Brain-Derived Neurotrophic Factor ,Brain ,Rats ,Mice, Inbred C57BL ,Cysteine Endopeptidases ,Neuroprotective Agents ,biology.protein ,Phosphorylation ,Amyloid precursor protein secretase ,Signal Transduction - Abstract
Alzheimer's disease (AD) is the most common dementia, and no disease-modifying therapeutic agents are currently available. BDNF/TrkB signaling is impaired in AD and is associated with prominent delta-secretase (δ-secretase, also known as asparaginyl endopeptidase or legumain) activation, which simultaneously cleaves both APP and Tau and promotes Aβ production and neurofibrillary tangles (NFT) pathologies. Here we show that the optimized δ-secretase inhibitor (#11a) or TrkB receptor agonist (CF3CN) robustly blocks δ-secretase activity separately, and their combination synergistically blunts δ-secretase, exhibiting promising therapeutic efficacy in 3xTg AD mouse model. The optimal δ-secretase inhibitor reveals demonstrable brain exposure and oral bioavailability, suppressing APP N585 and Tau N368 cleavage by δ-secretase. Strikingly, CF3CN treatment evidently escalates BDNF levels. Both #11a and CF3CN display strong in vivo PK/PD properties and ability to suppress δ-secretase activity in the brain. Orally administrated CF3CN strongly activates TrkB that triggers active Akt to phosphorylate δ-secretase T322, preventing its proteolytic activation and mitigating AD pathologies. #11a or CF3CN significantly diminishes AD pathogenesis and improves cognitive functions with the combination exhibiting the maximal effect. Thus, our data support that these derivatives are strong pharmaceutical candidates for the treatment of AD. more...
- Published
- 2021
- Full Text
- View/download PDF
28. Cathepsin S Evokes PAR2-Dependent Pain in Oral Squamous Cell Carcinoma Patients and Preclinical Mouse Models
- Author
-
Rocco Latorre, John C. Dolan, Brian L. Schmidt, Caroline M. Sawicki, Dong H. Kim, Nicole N. Scheff, Kenji Inoue, Malvin N. Janal, Nigel W. Bunnett, Nguyen Huu Tu, Dane D. Jensen, Bethany M. Anderson, Lei Yang, Hung D. Tran, Laura E. Edgington-Mitchell, Elyssa Chen, Robel G. Alemu, and Cheng Z. Liu more...
- Subjects
cancer pain ,Cancer Research ,Angiogenesis ,Inflammation ,03 medical and health sciences ,0302 clinical medicine ,Carcinoma ,Medicine ,pain ,PAR2 ,Receptor ,RC254-282 ,Protease-activated receptor 2 ,030304 developmental biology ,Cathepsin S ,0303 health sciences ,protease-activated receptor-2 ,business.industry ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Cancer ,oral cancer ,medicine.disease ,3. Good health ,oral squamous cell carcinoma ,stomatognathic diseases ,Allodynia ,Oncology ,cathepsin S ,Cancer research ,medicine.symptom ,business ,030217 neurology & neurosurgery - Abstract
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. more...
- Published
- 2021
- Full Text
- View/download PDF
29. Fluorescent diphenylphosphonate-based probes for detection of serine protease activity during inflammation
- Author
-
Laura E. Edgington-Mitchell, Nicholas Barlow, Aminath Samha, Luigi Aurelio, Nigel W. Bunnett, Monika Szabo, and Bim Graham
- Subjects
0301 basic medicine ,Proteases ,medicine.medical_treatment ,Clinical Biochemistry ,Organophosphonates ,Pharmaceutical Science ,Biochemistry ,Article ,law.invention ,Serine ,Mice ,03 medical and health sciences ,law ,Drug Discovery ,medicine ,Animals ,Molecular Biology ,Fluorescent Dyes ,Inflammation ,chemistry.chemical_classification ,Protease ,Molecular Structure ,biology ,Organic Chemistry ,Elastase ,Colitis ,Trypsin ,Molecular biology ,030104 developmental biology ,Enzyme ,Pancreatitis ,chemistry ,Neutrophil elastase ,biology.protein ,Recombinant DNA ,Molecular Medicine ,Serine Proteases ,medicine.drug - Abstract
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. more...
- Published
- 2017
- Full Text
- View/download PDF
30. Loss of O -Linked Protein Glycosylation in Burkholderia cenocepacia Impairs Biofilm Formation and Siderophore Activity and Alters Transcriptional Regulators
- Author
-
Hayley J. Newton, Elizabeth L. Hartland, Richard A. Strugnell, Clare V. Oates, Laura E. Edgington-Mitchell, Miku Kuba, Nichollas E. Scott, Cameron C. Oppy, Leila Jebeli, and Miguel A. Valvano
- Subjects
Molecular Biology and Physiology ,animal structures ,Glycosylation ,glycosylation ,Burkholderia cenocepacia ,lcsh:QR1-502 ,Siderophores ,macromolecular substances ,Proteomics ,Microbiology ,lcsh:Microbiology ,protein modification ,03 medical and health sciences ,chemistry.chemical_compound ,proteomics ,N-linked glycosylation ,Genes, Reporter ,DNA binding ,CepR ,Molecular Biology ,glycoproteins ,030304 developmental biology ,2. Zero hunger ,Regulation of gene expression ,chemistry.chemical_classification ,0303 health sciences ,biology ,030306 microbiology ,Chemistry ,Gene Expression Profiling ,pathogenesis ,Gene Expression Regulation, Bacterial ,biology.organism_classification ,QR1-502 ,Cell biology ,carbohydrates (lipids) ,Burkholderia ,Biofilms ,Proteome ,posttranslational modifications ,lipids (amino acids, peptides, and proteins) ,Glycoprotein ,Research Article ,Transcription Factors - Abstract
Protein glycosylation is increasingly recognized as a common posttranslational protein modification in bacterial species. Despite this commonality, our understanding of the role of most glycosylation systems in bacterial physiology and pathogenesis is incomplete. In this work, we investigated the effect of the disruption of O-linked glycosylation in the opportunistic pathogen Burkholderia cenocepacia using a combination of proteomic, molecular, and phenotypic assays. We find that in contrast to recent findings on the N-linked glycosylation systems of Campylobacter jejuni, O-linked glycosylation does not appear to play a role in proteome stabilization of most glycoproteins. Our results reveal that loss of glycosylation in B. cenocepacia strains leads to global proteome and transcriptional changes, including the repression of the quorum-sensing regulator cepR (BCAM1868) gene. These alterations lead to dramatic phenotypic changes in glycosylation-null strains, which are paralleled by both global proteomic and transcriptional alterations, which do not appear to directly result from the loss of glycosylation per se. This research unravels the pleiotropic effects of O-linked glycosylation in B. cenocepacia, demonstrating that its loss does not simply affect the stability of the glycoproteome, but also interferes with transcription and the broader proteome., O-linked protein glycosylation is a conserved feature of the Burkholderia genus. The addition of the trisaccharide β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc to membrane exported proteins in Burkholderia cenocepacia is required for bacterial fitness and resistance to environmental stress. However, the underlying causes of the defects observed in the absence of glycosylation are unclear. Using proteomics, luciferase reporter assays, and DNA cross-linking, we demonstrate the loss of glycosylation leads to changes in transcriptional regulation of multiple proteins, including the repression of the master quorum CepR/I. These proteomic and transcriptional alterations lead to the abolition of biofilm formation and defects in siderophore activity. Surprisingly, the abundance of most of the known glycosylated proteins did not significantly change in the glycosylation-defective mutants, except for BCAL1086 and BCAL2974, which were found in reduced amounts, suggesting they could be degraded. However, the loss of these two proteins was not responsible for driving the proteomic alterations, biofilm formation, or siderophore activity. Together, our results show that loss of glycosylation in B. cenocepacia results in a global cell reprogramming via alteration of the transcriptional regulatory systems, which cannot be explained by the abundance changes in known B. cenocepacia glycoproteins. IMPORTANCE Protein glycosylation is increasingly recognized as a common posttranslational protein modification in bacterial species. Despite this commonality, our understanding of the role of most glycosylation systems in bacterial physiology and pathogenesis is incomplete. In this work, we investigated the effect of the disruption of O-linked glycosylation in the opportunistic pathogen Burkholderia cenocepacia using a combination of proteomic, molecular, and phenotypic assays. We find that in contrast to recent findings on the N-linked glycosylation systems of Campylobacter jejuni, O-linked glycosylation does not appear to play a role in proteome stabilization of most glycoproteins. Our results reveal that loss of glycosylation in B. cenocepacia strains leads to global proteome and transcriptional changes, including the repression of the quorum-sensing regulator cepR (BCAM1868) gene. These alterations lead to dramatic phenotypic changes in glycosylation-null strains, which are paralleled by both global proteomic and transcriptional alterations, which do not appear to directly result from the loss of glycosylation per se. This research unravels the pleiotropic effects of O-linked glycosylation in B. cenocepacia, demonstrating that its loss does not simply affect the stability of the glycoproteome, but also interferes with transcription and the broader proteome. more...
- Published
- 2019
- Full Text
- View/download PDF
31. Tissue-ABPP enables high-resolution confocal fluorescence imaging of serine hydrolase activity in cryosections – Application to glioma brain unveils activity hotspots originating from tumor-associated neutrophils
- Author
-
Haritha Samaranayake, Marcin Drag, Sanna Pasonen-Seppänen, Sara Kälvälä, Laura E. Edgington-Mitchell, Jarmo T. Laitinen, Eemeli Moisio, Markku Varjosalo, Prosanta K. Singha, Paulina Kasperkiewicz, Juha R. Savinainen, Hermina Jakupović, Niina Aaltonen, Kirsi Rilla, and Thomas Wirth more...
- Subjects
Streptavidin ,0303 health sciences ,Fluorescence-lifetime imaging microscopy ,Confocal ,medicine.disease ,3. Good health ,Cell biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Biotin ,chemistry ,030220 oncology & carcinogenesis ,Glioma ,Proteome ,medicine ,Immunohistochemistry ,Serine hydrolase activity ,030304 developmental biology - Abstract
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. The ABPP approach 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. Here, we advance the ABPP methodology to glioma brain cryosections, enabling high-resolution confocal fluorescence imaging of SH activity in different cell types of the tumor microenvironment, identified by using extensive immunohistochemistry on activity probe labeled sections. We name this technique tissue-ABPP to distinguish it from conventional gel-based ABPP. We show heightened SH activity in glioma vs. normal brain and unveil activity hotspots originating from tumor-associated neutrophils. Thorough optimization and validation is provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. Tissue-ABPP enables a wide range of applications for confocal imaging of SH activity in any type of tissue or animal species. more...
- Published
- 2019
- Full Text
- View/download PDF
32. Proteomics and Imaging in Crohn's Disease: TAILS of Unlikely Allies
- Author
-
B Mainoli, Simon A. Hirota, Laura E. Edgington-Mitchell, Cathy Lu, and Antoine Dufour
- Subjects
0301 basic medicine ,Diagnostic Imaging ,Proteomics ,medicine.medical_specialty ,Abdominal pain ,Disease ,Toxicology ,Inflammatory bowel disease ,Pathogenesis ,03 medical and health sciences ,0302 clinical medicine ,Crohn Disease ,Medical imaging ,medicine ,Humans ,Intensive care medicine ,Pharmacology ,Crohn's disease ,business.industry ,Precision medicine ,medicine.disease ,Inflammatory Bowel Diseases ,3. Good health ,030104 developmental biology ,Etiology ,medicine.symptom ,business ,030217 neurology & neurosurgery ,Biomarkers - Abstract
Crohn's disease (CD) is a chronic relapsing inflammatory bowel disease (IBD) that may be marked by debilitating symptoms of abdominal pain and obstruction. The etiology and pathogenesis of the disease are not fully understood, and treatment with corticosteroids, biologics, and surgical intervention are the usual therapeutic options. Diagnosis, disease activity, and therapeutic response are currently assessed by endoscopy, cross-sectional imaging, and biomarkers. However, challenges remain regarding the efficacy of the drugs and safety of these imaging techniques. There are also limitations with current clinical and laboratory tools for diagnosis, disease progression, and treatment response. Here, we discuss how the integration of proteomics and activity-based probes, along with intestinal ultrasound, an easily repeatable and well-tolerated diagnostic imaging modality, can address these challenges and may provide a novel precision medicine-based approach for the treatment of CD. more...
- Published
- 2019
33. Application of a chemical probe to detect neutrophil elastase activation during inflammatory bowel disease
- Author
-
Markus Fleischmann, Nigel W. Bunnett, Paulina Kasperkiewicz, Luigi Aurelio, Celine Morissette, Marcin Drag, Daniel P. Poole, Bethany M. Anderson, Ian R. van Driel, Laura E. Edgington-Mitchell, Garrett Z. Ng, Stephen Vanner, and Brian L. Schmidt more...
- Subjects
Male ,0301 basic medicine ,Proteases ,Neutrophils ,medicine.medical_treatment ,Mice, Nude ,lcsh:Medicine ,Inflammatory bowel disease ,Article ,Neutrophil Activation ,Legionella pneumophila ,Pathogenesis ,Mice ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Animals ,Humans ,Colitis ,lcsh:Science ,Cells, Cultured ,Serine protease ,Mouth neoplasm ,Mice, Inbred BALB C ,Multidisciplinary ,Protease ,biology ,Chemistry ,lcsh:R ,Diagnostic markers ,Chronic inflammation ,medicine.disease ,Molecular biology ,3. Good health ,Mice, Inbred C57BL ,030104 developmental biology ,Ulcerative colitis ,030220 oncology & carcinogenesis ,Neutrophil elastase ,biology.protein ,Colitis, Ulcerative ,Female ,Mouth Neoplasms ,lcsh:Q ,Legionnaires' Disease ,Leukocyte Elastase - Abstract
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. more...
- Published
- 2019
- Full Text
- View/download PDF
34. Loss ofO-linked protein glycosylation inBurkholderia cenocepaciaimpairs biofilm formation and siderophore production via alteration of quorum sensing regulation
- Author
-
Hayley J. Newton, Elizabeth L. Hartland, Richard A. Strugnell, Miguel A. Valvano, Clare V. Oates, Nichollas E. Scott, Cameron C. Oppy, Leila Jebeli, Miku Kuba, and Laura E. Edgington-Mitchell
- Subjects
chemistry.chemical_classification ,0303 health sciences ,Glycosylation ,biology ,Burkholderia cenocepacia ,030306 microbiology ,Virulence ,biology.organism_classification ,Cell biology ,carbohydrates (lipids) ,03 medical and health sciences ,chemistry.chemical_compound ,Quorum sensing ,Burkholderia ,Regulon ,chemistry ,Proteome ,lipids (amino acids, peptides, and proteins) ,Glycoprotein ,030304 developmental biology - Abstract
O-linked protein glycosylation is a conserved feature of theBurkholderiagenus. ForBurkholderia cenocepacia, the addition of the trisaccharide β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc to membrane exported proteins is required for virulence and resistance to environmental stress. However, the underlying causes of the defects observed in the absence of glycosylation are unclear. This study demonstrates that the globalB. cenocepaciaproteome undergoes dramatic changes consistent with alterations in global transcriptional regulation in the absence of glycosylation. Using luciferase reporter assays and DNA cross-linking analysis, we confirm the repression of the master quorum sensing regulon CepR/I in response to the loss of glycosylation, which leads to the abolition of biofilm formation, defects in siderophore production, and reduced virulence. The abundance of most of the known glycosylated proteins did not significantly change in the glycosylation-defective mutants except for BCAL1086 and BCAL2974, which were found in reduced amount, suggesting they could be degraded. However, the loss of these two proteins was not responsible for driving the proteomic alterations, as well as for reduced virulence and siderophore production. Together, our results show that loss of glycosylation inB. cenocepaciaresults in a global cell reprogramming via alteration of the CepR/I regulon, which cannot be explained by the abundance changes in knownB. cenocepaciaglycoproteins.IMPORTANCEProtein glycosylation is increasingly recognised as a common protein modification in bacterial species. Despite this commonality our understanding of the role of most glycosylation systems in bacterial physiology and pathogenesis is incomplete. In this work, we investigated the effect of the disruption ofO-linked glycosylation in the opportunistic pathogenBurkholderia cenocepaciausing a combination of proteomic, molecular and phenotypic assays. We find that in contrast to recent findings on theN-linked glycosylation systems ofCampylobacter jejuni, O-linked glycosylation does not appear to play a role in proteome stabilization of most glycoproteins. Our results reveal that virulence attenuation observed within glycosylation-nullB. cenocepaciastrains are consistent with alteration of the master virulence regulator CepR. The repression of CepR transcription and its associated phenotypes support a model in which the virulence defects observed in glycosylation-null strains are at least in part due to transcriptional alteration and not the direct result of the loss of glycosylationper-se. This research unravels the pleotropic effects ofO-linked glycosylation inB. cenocepacia,demonstrating that its loss does not simply affect the stability of the glycoproteome, but also interferes with transcription and the broader proteome. more...
- Published
- 2019
- Full Text
- View/download PDF
35. Sez6 levels are elevated in cerebrospinal fluid of patients with inflammatory pain–associated conditions
- Author
-
James Ziogas, Paul S. Myles, Laura E. Edgington-Mitchell, Jon E. Mangum, Alexios A. Adamides, Jenny M. Gunnersen, Maria Roitman, Nigel W. Bunnett, and Hearan Choo-Bunnett
- Subjects
medicine.medical_specialty ,Inflammatory pain ,Central nervous system ,CSF ,Chronic pain ,02 engineering and technology ,01 natural sciences ,Gastroenterology ,lcsh:RD78.3-87.3 ,Cerebrospinal fluid ,Basic Science ,Western blot ,Internal medicine ,0103 physical sciences ,0202 electrical engineering, electronic engineering, information engineering ,medicine ,010306 general physics ,medicine.diagnostic_test ,business.industry ,Brief Report ,Seizure-related protein 6 ,BACE1 ,medicine.disease ,3. Good health ,Blot ,Anesthesiology and Pain Medicine ,medicine.anatomical_structure ,lcsh:Anesthesiology ,Peripheral nerve injury ,Knockout mouse ,Neuropathic pain ,ComputingMethodologies_DOCUMENTANDTEXTPROCESSING ,020201 artificial intelligence & image processing ,business - Abstract
Supplemental Digital Content is Available in the Text., Introduction: Seizure-related protein 6 (Sez6) contributes to chronic pain development as sez6 knockout mice show attenuated pain behaviours after peripheral nerve injury, compared with control mice. The type I transmembrane isoform of Sez6 is cleaved by the β-amyloid precursor protein cleavage enzyme 1 (BACE1), resulting in Sez6 extracellular domain shedding from the neuron surface. Objectives: To determine whether this BACE1-shed form of Sez6 can be detected in the cerebrospinal fluid (CSF) and whether Sez6 levels in the CSF are altered in neuropathic pain or chronic inflammatory pain (IP). Methods: We analysed the CSF samples collected during surgery from patients with chronic neuropathic pain (n = 8) or IP (n = 33), comparing them to the CSF samples from patients with suspected subarachnoid haemorrhage that was subsequently excluded (nonsurgical group, n = 5). Western blots were used to determine the relative Sez6 levels in the CSF from the different patient and nonsurgical comparison groups. Results: The results show that BACE1-shed Sez6 can be readily detected in the CSF by Western blot and that the levels of Sez6 are significantly higher in the IP group than in the nonsurgical comparison group. Conclusion: The association between elevated Sez6 levels in the CSF and IP is further evidence for persistent alterations in central nervous system activity in chronic IP conditions. more...
- Published
- 2019
- Full Text
- View/download PDF
36. Antagonism of the proinflammatory and pronociceptive actions of canonical and biased agonists of protease‐activated receptor‐2
- Author
-
Tina Marie Lieu, Laura E. Edgington-Mitchell, Nigel W. Bunnett, David P. Fairlie, Romke Bron, Daniel P. Poole, Peter McLean, Nicholas Barlow, E. Savage, Peishen Zhao, and Rink-Jan Lohman
- Subjects
Male ,0301 basic medicine ,Proteases ,Administration, Oral ,Proinflammatory cytokine ,Rats, Sprague-Dawley ,Mice ,Structure-Activity Relationship ,03 medical and health sciences ,medicine ,Animals ,Receptor, PAR-2 ,Protease-activated receptor 2 ,Cathepsin S ,Inflammation ,Mice, Knockout ,Pharmacology ,Cathepsin ,Dose-Response Relationship, Drug ,Chemistry ,Elastase ,Nociceptors ,Research Papers ,Rats ,Cell biology ,Mice, Inbred C57BL ,030104 developmental biology ,Nociception ,Biochemistry ,Hyperalgesia ,medicine.symptom ,Oligopeptides ,Research Paper - Abstract
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 [Ca2+]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. more...
- Published
- 2016
- Full Text
- View/download PDF
37. Pathophysiological roles of proteases in gastrointestinal disease
- Author
-
Laura E. Edgington-Mitchell
- Subjects
0301 basic medicine ,Proteases ,Gastrointestinal Diseases ,Physiology ,Colorectal cancer ,medicine.medical_treatment ,Population ,Disease ,Biology ,Inflammatory bowel disease ,03 medical and health sciences ,Physiology (medical) ,medicine ,Animals ,Humans ,education ,Irritable bowel syndrome ,Cathepsin S ,education.field_of_study ,Protease ,Hepatology ,Gastroenterology ,medicine.disease ,030104 developmental biology ,Immunology ,Peptide Hydrolases - Abstract
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. more...
- Published
- 2016
- Full Text
- View/download PDF
38. System-wide biochemical analysis reveals ozonide antimalarials initially act by disrupting Plasmodium falciparum haemoglobin digestion
- Author
-
Bethany M. Anderson, Susan A. Charman, Darren J. Creek, Carlo Giannangelo, Amanda De Paoli, Ghizal Siddiqui, and Laura E. Edgington-Mitchell
- Subjects
Proteomics ,Plasmodium ,Erythrocytes ,Adamantane ,Biochemistry ,Hemoglobins ,chemistry.chemical_compound ,Drug Metabolism ,Heterocyclic Compounds ,Medicine and Health Sciences ,Biology (General) ,Artemisinin ,0303 health sciences ,biology ,030302 biochemistry & molecular biology ,Drugs ,Oxides ,Peroxides ,Chemistry ,Physical Sciences ,Metabolic Pathways ,Research Article ,medicine.drug ,Proteases ,QH301-705.5 ,Plasmodium falciparum ,Immunology ,Quantitative proteomics ,Microbiology ,Antimalarials ,Heterocyclic Compounds, 1-Ring ,03 medical and health sciences ,Piperaquine ,Virology ,Parasite Groups ,Parasitic Diseases ,Genetics ,medicine ,Humans ,Metabolomics ,Ozonide ,Spiro Compounds ,Pharmacokinetics ,Arterolane ,Mode of action ,Molecular Biology ,030304 developmental biology ,Pharmacology ,Catabolism ,Chemical Compounds ,Protein turnover ,Biology and Life Sciences ,RC581-607 ,biology.organism_classification ,Metabolism ,chemistry ,Parasitology ,Immunologic diseases. Allergy ,Apicomplexa - Abstract
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., Author summary The ozonides are a novel class of fully synthetic antimalarial drugs with potent activity against all parasite species that cause malaria, including the deadliest, Plasmodium falciparum. With the emergence of resistance to current frontline artemisinin-based antimalarials, new drugs are urgently needed and a clear understanding of their mechanism of action is essential so that they can be optimally deployed in the field. Here, we studied the biochemical effects of two ozonides, OZ277 (marketed in India in combination with piperaquine) and OZ439 (in Phase IIb clinical trials) in P. falciparum parasites using an untargeted multi-omics approach consisting of proteomics, peptidomics and time-dependent metabolomics, along with activity-based protease profiling. We found that the ozonides initially disrupt haemoglobin metabolism and that they likely engage the parasite proteostatic stress response. Furthermore, when the duration of ozonide exposure was extended beyond 3 hours to reflect clinically-relevant exposure periods, additional parasite biochemical pathways were perturbed. This comprehensive analysis provides new insight into the antimalarial mode of action of ozonides and provides new opportunities for interventions to enhance their antimalarial efficacy. more...
- Published
- 2020
- Full Text
- View/download PDF
39. Correction: Novel broad-spectrum activity-based probes to profile malarial cysteine proteases
- Author
-
Michele S. Y. Tan, Mateo I. Sánchez, Ambrosius P. Snijders, Edgar Deu, Laura E. Edgington-Mitchell, Dara Davison, Bethany M. Anderson, and Stephen H. Howell
- Subjects
Proteases ,Broad spectrum ,Multidisciplinary ,Chemistry ,Science ,Medicine ,Computational biology ,Cysteine - Abstract
[This corrects the article DOI: 10.1371/journal.pone.0227341.].
- Published
- 2020
- Full Text
- View/download PDF
40. PD-1 Inhibitory Receptor Downregulates Asparaginyl Endopeptidase and Maintains Foxp3 Transcription Factor Stability in Induced Regulatory T Cells
- Author
-
Shoba Amarnath, David Knight, Michael Eckhaus, Sanders I. van Kasteren, Ethan M. Shevach, Grace Mallett, Arunakumar Gangaplara, Lukasz P. Liniany, Francis A. Flomerfelt, Laura E. Edgington-Mitchell, Tania C. Felizardo, Colin Watts, Matthew Bogyo, Jinfang Zhu, Chaido Stathopoulou, Arian Laurence, Jonathan Martinez-Fabregas, Joshua J. Yim, Daniel H. Fowler, Rolando Berlinguer-Palmini, and Leigh Samsel more...
- Subjects
0301 basic medicine ,Tumor-infiltrating lymphocytes ,Cellular differentiation ,Immunology ,Cell ,FOXP3 ,chemical and pharmacologic phenomena ,Inflammation ,Biology ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Infectious Diseases ,medicine.anatomical_structure ,Immune system ,Antigen ,030220 oncology & carcinogenesis ,medicine ,Immunology and Allergy ,medicine.symptom ,Transcription factor - Abstract
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. more...
- Published
- 2018
41. Protease-activated receptor-2 in endosomes signals persistent pain of irritable bowel syndrome
- Author
-
Holly R. Yeatman, Stuart M. Brierley, Dane D. Jensen, Peishen Zhao, Aditi Bhattacharya, Bernard L. Flynn, Nigel W. Bunnett, Christopher J.H. Porter, Rocco Latorre, Elyssa Chen, Daniel P. Poole, Laura E. Edgington-Mitchell, Luke A. Pattison, Peter McLean, Tina Marie Lieu, Michelle L. Halls, Nicholas A. Veldhuis, Nicole N. Scheff, Brian L. Schmidt, Giang Thanh Le, Gareth A. Hicks, Meritxell Canals, Stephen Vanner, Nestor N. Jiménez-Vargas, Luigi Aurelio, Joel Castro, and Carmen Klein Herenbrink more...
- Subjects
0301 basic medicine ,Nociception ,Endosome ,Endosomes ,Pharmacology ,Endocytosis ,Irritable Bowel Syndrome ,03 medical and health sciences ,medicine ,Animals ,Humans ,Receptor, PAR-2 ,Trypsin ,Extracellular Signal-Regulated MAP Kinases ,Protease-activated receptor 2 ,Cathepsin S ,Cathepsin ,Multidisciplinary ,Chemistry ,Beta-Arrestins ,Nociceptors ,3. Good health ,030104 developmental biology ,Allodynia ,PNAS Plus ,medicine.symptom ,Chronic Pain ,Signal Transduction - Abstract
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. more...
- Published
- 2018
42. Myoepithelial cell-specific expression of stefin A as a suppressor of early breast cancer invasion
- Author
-
Min Hu, Belinda S. Parker, Natasha K Brockwell, Anne Holliday, Kornelia Polyak, C.I. Selinger, Alex Spurling, Sandra A O'Toole, Maoshan Chen, Hendrika M. Duivenvoorden, Timothy J. Molloy, Cameron J. Nowell, Kara L. Britt, David W. Greening, David A. Stroud, Jai Rautela, Elizabeth Robbins, Bonnie F. Sloane, Cheok Soon Lee, Andreas Möller, Matthew Bogyo, and Laura E. Edgington-Mitchell more...
- Subjects
0301 basic medicine ,Pathology ,medicine.medical_specialty ,Antineoplastic Agents ,Breast Neoplasms ,Biology ,Cysteine Proteinase Inhibitors ,medicine.disease_cause ,Transfection ,Cathepsin B ,Pathology and Forensic Medicine ,03 medical and health sciences ,Mice ,Breast cancer ,Mammary Glands, Animal ,Cell Movement ,Cell Line, Tumor ,medicine ,Biomarkers, Tumor ,Tumor Microenvironment ,Animals ,Humans ,Cystatin A ,Neoplasm Invasiveness ,skin and connective tissue diseases ,Mammary Glands, Human ,Uncategorized ,Tumor microenvironment ,Carcinoma in situ ,Tumor Suppressor Proteins ,Myoepithelial cell ,Epithelial Cells ,Ductal carcinoma ,medicine.disease ,Coculture Techniques ,030104 developmental biology ,Carcinoma, Intraductal, Noninfiltrating ,Female ,RNA Interference ,Carcinogenesis ,Signal Transduction - Abstract
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. more...
- Published
- 2017
43. Live Cell Imaging and Profiling of Cysteine Cathepsin Activity Using a Quenched Activity-Based Probe
- Author
-
Laura E, Edgington-Mitchell, Matthew, Bogyo, and Martijn, Verdoes
- Subjects
Microscopy, Fluorescence ,Animals ,Electrophoresis, Polyacrylamide Gel ,Cysteine ,Cathepsins ,Fluorescent Dyes - Abstract
Since protease activity is highly regulated by structural and environmental influences, the abundance of a protease often does not directly correlate with its activity. Because in most of the cases it is the activity of a protease that gives rise to its biological relevance, tools to report on this activity are of great value to the research community. Activity-based probes (ABPs) are small molecule tools that allow for the monitoring and profiling of protease activities in complex biological systems. The class of fluorescent quenched ABPs (qABPs), being intrinsically "dark" and only emitting fluorescence after reaction with the target protease, are ideally suited for imaging techniques such as small animal noninvasive fluorescence imaging and live cell fluorescence microscopy. An additional powerful characteristic of qABPs is their covalent and irreversible modification of the labeled protease, enabling in-depth target characterization. Here we describe the synthesis of a pan-cysteine cathepsin qABP BMV109 and the application of this probe to live cell fluorescence imaging and fluorescent SDS-PAGE cysteine cathepsin activity profiling. more...
- Published
- 2016
44. Live Cell Imaging and Profiling of Cysteine Cathepsin Activity Using a Quenched Activity-Based Probe
- Author
-
Martijn Verdoes, Matthew Bogyo, and Laura E. Edgington-Mitchell
- Subjects
0301 basic medicine ,Cathepsin ,Fluorescence-lifetime imaging microscopy ,Protease ,medicine.medical_treatment ,Biology ,010402 general chemistry ,01 natural sciences ,Small molecule ,Fluorescence ,0104 chemical sciences ,03 medical and health sciences ,030104 developmental biology ,Biochemistry ,Live cell imaging ,Fluorescence microscope ,medicine ,Cysteine - Abstract
Since protease activity is highly regulated by structural and environmental influences, the abundance of a protease often does not directly correlate with its activity. Because in most of the cases it is the activity of a protease that gives rise to its biological relevance, tools to report on this activity are of great value to the research community. Activity-based probes (ABPs) are small molecule tools that allow for the monitoring and profiling of protease activities in complex biological systems. The class of fluorescent quenched ABPs (qABPs), being intrinsically "dark" and only emitting fluorescence after reaction with the target protease, are ideally suited for imaging techniques such as small animal noninvasive fluorescence imaging and live cell fluorescence microscopy. An additional powerful characteristic of qABPs is their covalent and irreversible modification of the labeled protease, enabling in-depth target characterization. Here we describe the synthesis of a pan-cysteine cathepsin qABP BMV109 and the application of this probe to live cell fluorescence imaging and fluorescent SDS-PAGE cysteine cathepsin activity profiling. more...
- Published
- 2016
- Full Text
- View/download PDF
45. Detection of Active Caspases During Apoptosis Using Fluorescent Activity-Based Probes
- Author
-
Matthew Bogyo and Laura E. Edgington-Mitchell
- Subjects
0301 basic medicine ,Proteases ,Fluorophore ,genetic structures ,medicine.medical_treatment ,Apoptosis ,Bioinformatics ,Article ,Mice ,03 medical and health sciences ,Enzyme activator ,chemistry.chemical_compound ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,Caspase ,Fluorescent Dyes ,Protease ,Staining and Labeling ,biology ,Chemistry ,Xenograft Model Antitumor Assays ,Fluorescence ,In vitro ,Molecular Imaging ,Enzyme Activation ,030104 developmental biology ,Biochemistry ,Caspases ,Colonic Neoplasms ,biology.protein ,Electrophoresis, Polyacrylamide Gel - Abstract
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. more...
- Published
- 2016
- Full Text
- View/download PDF
46. Legumain is activated in macrophages during pancreatitis
- Author
-
Wouter A. van der Linden, Luigi Aurelio, Walter Halangk, Alicia K. Fleming, Matthew Bogyo, Peter Storz, Thomas Wartmann, Vasilena Gocheva, Martijn Verdoes, Tina Marie Lieu, Daniel Edgington-Mitchell, Nimali P. Withana, Laura E. Edgington-Mitchell, Belinda S. Parker, Bim Graham, John B. Furness, Johanna A. Joyce, Nigel W. Bunnett, and Thomas Reinheckel more...
- Subjects
0301 basic medicine ,Pathology ,medicine.medical_specialty ,Physiology ,Cancer development and immune defence Radboud Institute for Molecular Life Sciences [Radboudumc 2] ,Inflammation ,Legumain ,Cathepsin B ,Gene Expression Regulation, Enzymologic ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Physiology (medical) ,Pancreatic cancer ,medicine ,Animals ,Trypsinogen activation ,Enzyme Inhibitors ,Ceruletide ,Mice, Knockout ,Hepatology ,biology ,Macrophages ,Gastroenterology ,Pancreatic Physiology/Pathophysiology ,medicine.disease ,Mice, Inbred C57BL ,Cysteine Endopeptidases ,030104 developmental biology ,medicine.anatomical_structure ,Pancreatitis ,030220 oncology & carcinogenesis ,biology.protein ,Female ,medicine.symptom ,Pancreas - Abstract
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. more...
- Published
- 2016
- Full Text
- View/download PDF
47. Demonstration of elevated levels of active cathepsin S in dextran sulfate sodium colitis using a new activatable probe
- Author
-
Raquel Guerrero-Alba, Joshua W. Conner, Daniel P. Poole, Yasmin Nasser, Tina Marie Lieu, Nicholas Barlow, Peishen Zhao, Stephanie Vanner, Bimbil Graham, Laura E. Edgington-Mitchell, Namit Sharma, Nicholas A. Veldhuis, Erik Lindström, Andrew W.B. Craig, and Nigel W. Bunnett more...
- Subjects
Proteases ,Physiology ,medicine.medical_treatment ,Inflammation ,Mice ,medicine ,Animals ,Colitis ,Receptor ,Cathepsin S ,Fluorescent Dyes ,Cathepsin ,Gastrointestinal tract ,Protease ,Endocrine and Autonomic Systems ,Chemistry ,Dextran Sulfate ,Gastroenterology ,medicine.disease ,Molecular biology ,Cathepsins ,Mice, Inbred C57BL ,Disease Models, Animal ,Biochemistry ,medicine.symptom - Abstract
Background Proteases play a major role in inflammatory diseases of the gastrointestinal tract. Activatable probes are a major technological advance, enabling sensitive detection of active proteases in tissue samples. Our aim was to synthesize an activatable probe for cathepsin S and validate its use in a mouse model of colitis. Methods We designed and synthesized a new fluorescent activatable probe, NB200, for the detection of active cathepsin S. Colitis was induced in C57BL/6 mice by the administration of 3% dextran sulfate sodium (DSS). Homogenized mouse colons, with or without the addition of the specific cathepsin S inhibitor MV026031, were incubated with NB200 in a fluorescent plate reader. Key Results NB200 selectively detected purified cathepsin S and not other common inflammatory proteases. Homogenates of colon from mice with DSS colitis induced a significant fluorescent increase when compared to control animals (control vs DSS: p more...
- Published
- 2015
48. Long noncoding RNAs: novel links to inflammatory bowel disease?
- Author
-
Laura E. Edgington-Mitchell
- Subjects
0301 basic medicine ,Physiology ,Disease ,Bioinformatics ,Inflammatory bowel disease ,03 medical and health sciences ,Physiology (medical) ,Humans ,Medicine ,Regulation of gene expression ,Hepatology ,business.industry ,Incidence (epidemiology) ,Gastroenterology ,Inflammatory Bowel Diseases ,medicine.disease ,Ulcerative colitis ,digestive system diseases ,030104 developmental biology ,Gene Expression Regulation ,Etiology ,Cytokines ,RNA, Long Noncoding ,business ,Biomarkers - Abstract
inflammatory bowel diseases (IBDs), comprising ulcerative colitis and Crohn's 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 more...
- Published
- 2016
- Full Text
- View/download PDF
49. Probes to monitor activity of the paracaspase MALT1
- Author
-
Matthew Bogyo, Guy S. Salvesen, Laura E. Sanman, Laura E. Edgington-Mitchell, Marcin Poreba, Janna Hachmann, and Marcin Drag
- Subjects
Scaffold protein ,Clinical Biochemistry ,Plasma protein binding ,Transfection ,Biochemistry ,Article ,03 medical and health sciences ,0302 clinical medicine ,Cell Line, Tumor ,Drug Discovery ,Humans ,Molecular Biology ,Caspase ,030304 developmental biology ,Pharmacology ,0303 health sciences ,biology ,HEK 293 cells ,NF-kappa B ,General Medicine ,Paracaspase ,NFKB1 ,Recombinant Proteins ,Neoplasm Proteins ,MALT1 ,Kinetics ,HEK293 Cells ,Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein ,030220 oncology & carcinogenesis ,Caspases ,Molecular Probes ,Mutation ,Proteolysis ,biology.protein ,Molecular Medicine ,Signal transduction ,Protein Binding ,Signal Transduction - Abstract
SummaryThe 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. more...
- Published
- 2014
50. Tu1879 Legumain Is a Novel Biomarker and Therapeutic Target in Inflammatory Bowel Disease
- Author
-
Daniel P. Poole, Simona E. Carbone, Nigel W. Bunnett, and Laura E. Edgington-Mitchell
- Subjects
Hepatology ,biology ,business.industry ,Gastroenterology ,Cancer research ,biology.protein ,Biomarker (medicine) ,Medicine ,Legumain ,business ,medicine.disease ,Inflammatory bowel disease - Published
- 2016
- Full Text
- View/download PDF
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.