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Principal Investigator: THOMAS H BUGGE
Organization: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
Fiscal Year: 2020
Award: $2,244,204
Funding agency: National Institute of Dental and Craniofacial Research
The overall aim of this project is to understand the biochemistry, biology, and pathology of cell surface-associated proteolysis, with an emphasis on determining the contribution to the development, homeostasis, regeneration, and malignant transformation of oral tissues.
Cell surface serine proteases as regulators of epithelial development, homeostasis regeneration, and malignancy:
Background: Cell behavior is regulated by a vast number of proteases and protease inhibitors that function in the pericellular environment to provide focal proteolysis essential for cytokine/growth factor maturation, matrix remodeling, signaling receptor activation and shedding, ion channel activity, and more. We are continuing our efforts to understand the molecular functions of membrane-anchored serine proteases and their inhibitors in vertebrate development, epithelial homeostasis, and epithelial carcinogenesis by using a combined biochemical, cell biological, and genetic approach.
Iterative, multiplexed CRISPR-mediated gene editing for functional analysis of complex protease gene clusters
Research accomplished:
The HAT/DESC cluster encodes seven closely related membrane-anchored serine proteases. These proteases display a coordinated expression in squamous epithelium of the oral cavity, upper airways, and epidermis. Little is known about the physiological functions of this protease family. The high amino acid identity and overlapping expression pattern of HAT/DESC proteases predict extensive redundancy, and the tight clustering of the genes encoding these proteases has complicated traditional loss-of-function genetics. We used CRISPR-mediated gene editing to generate 18 unique congenic mouse strains lacking combinations of HAT/DESC proteases. This included a mouse strain deficient in all seven HAT/DESC proteases. Surprisingly, mice lacking all HAT/DESC proteases developed to term, were healthy and fertile, and displayed only a modest impairment of epidermal barrier function, which was exclusively due to loss of HAT-like 4. Thus, mice lacking all HAT/DESC proteases displayed transepidermal water loss rates identical to mice lacking only HAT-like 4, and, conversely, mice lacking all HAT/DESC proteases, except HAT-like 4, displayed transepidermal water loss rates identical to wildtype mice. The study yields novel insights into the function of HAT/DESC proteases and provides multiple new mouse strains for functional studies of this protease sub-family in physiological and in pathophysiological processes.
Extracellular matrix degradation in physiological and pathophysiological processes:
Background: Research performed within the last five decades led to the identification and extensive characterization of extracellular matrix (ECM)-degrading enzymes. However, the cellular orchestration of ECM degradation and the contribution of aberrant ECM turnover pathways to human disease is still incompletely understood.
Fibrin-driven alveolar bone destruction in periodontitis
Fibrin is a provisional extracellular matrix protein that is formed by polymerization of thrombin-cleaved fibrinogen and cross-linking of the polymer by factor XIII. Fibrin is highly proinflammatory, and, unless removed in a timely manner, causes chronic inflammation and tissue damage by mechanisms not yet completely understood. Humans and mice with compromised fibrinolytic function display excess gingival fibrin deposition and severe periodontal disease. Fibrinolysis is achieved by the proteolytic conversion of the inactive zymogen, plasminogen (Plg), to the active protease, plasmin, by the serine proteases. We found that mice with severe defects in fibrinolysis all displayed spontaneous oral inflammation and severe periodontal bone destruction. Examination of the inflamed periodontal tissue revealed a significant neutrophil/monocyte infiltrate. Interestingly, homozygous knock-in fibrinogen-g390-396A mice, which express a mutant form of fibrinogen lacking the aMb2 myeloid integrin binding site, displayed reduced periodontal bone resorption when compared to their wildtype littermates. Moreover, periodontal bone destruction was completely reversed by superimposing either complete fibrinogen-deficiency or by expression of the mutant fibrinogen-g390-396A. Surprisingly, although not developing immunopathology, Plg-deficient mice expressing the mutant fibrinogen-g390-396A presented with gingival fibrin deposition and inflammatory cell accumulation comparable to Plg-deficient littermates expressing wildtype fibrinogen, suggesting that the local myeloid cell engagement of fibrin through aMb2 is essential for the initiation of periodontal bone destruction. In collaboration with Andrew D. Doyle, NIC, we proceeded to develop new assays to study neutrophil effector functions in vitro, By applying these assays, we found that neutrophils plated on mutant fibrinogen-g390-396A showed significantly decreased adhesion, reactive oxygen species (ROS) production and neutrophil extracellular trap formation (NETosis) compared to neutrophils plated on wildtype fibrin, indicating that fibrin-neutrophil interaction through the fibrin aMb2-binding domain leads to neutrophil activation. Furthermore, blunting neutrophil effector functions in vivo by genetic elimination of neutrophil elastase or by systemic treatment with DNAse I alleviated periodontal disease in Plg-deficient mice. Collectively, these data demonstrate that the local engagement of fibrin by neutrophils via fibrin-aMb2 ligation induces periodontal bone loss by triggering key neutrophil effector functions.
Reengineered bacterial cytotoxins as anti-tumor and protease imaging agents:
Background: Elevated expression of matrix-degrading proteases is a hallmark of malignancy. We are engaged in a long-standing collaboration with Steve Leppla, NIAID, on the development of reengineered bacterial cytotoxins activated by proteases expressed in the tumor microenvironment, as novel therapeutic agents for cancer and as tools for the imaging of specific cell surface proteolytic activity.
Visualizing CMG2- and TEM8-dependent anthrax toxin intoxication
Anthrax lethal toxin and anthrax edema toxin are binary toxins consisting of a common cell binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). Intoxication entails the binding of PA to either of two cell surface receptors, Capillary Morphogenesis Protein 2 (CMG2) or Tumor Endothelial Marker 8 (TEM8), followed by the cleavage of the receptor-bound PA by furin or furin-like proprotein convertases, which enables the binding, endocytosis, and cytoplasmic translocation of LF and EF. Controversy exists as to the cellular location of functional CMG2 and TEM8 receptors, making an assay for direct imaging of anthrax intoxication desirable. To develop such and assay, we generated a chimeric protein consisting of the N-terminal domain of LF fused to a nuclear localization signal-tagged Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were co-administered to transgenic mT/mG mice that express a membrane-tagged red fluorescent protein in the absence of Cre activity and a membrane-tagged green fluorescent protein in the presence of Cre activity, cellular intoxication could be directly visualized at single cell resolution. We used the newly generated assay to directly visualize CMG2- and TEM8-dependent anthrax toxin intoxication. For this purpose, we bred mT/mG mice to CMG2 and TEM8-deficient mice to generate mT/mG mice deficient in either of the two anthrax toxin receptors. Challenging these mice with PA and LFn-NLS-Cre revealed that CMG2 is essential for intoxication of liver and heart while loss of TEM8 partially prevented intoxication of kidney and spleen.
Terms: <ATR receptor><Active Oxygen><Adhesions><Afibrinogenemia><Alveolar Bone Loss><Alveolar Resorption><Amino Acids><Anthrax><Anthrax disease><Antigens><Antiproteases><Assay><Autoregulation><B anthracis LeTx><B anthracis LeTx toxin><Bacillus anthracis LF protein><Bacillus anthracis lef protein><Bacillus anthracis lethal toxin><Binding><Binding Sites><Bioassay><Biochemical><Biochemistry><Biologic Assays><Biological><Biological Assay><Biological Chemistry><Biology><Blood Coagulation Factor I><Blood Coagulation Factor One><Blood Coagulation Factor XIII><Blood Factor One><Blood Neutrophil><Blood Polymorphonuclear Neutrophil><Blood capillaries><Blood monocyte><Body Tissues><Bone Resorption><CRE Recombinase><CRISPR><CRISPR/Cas system><Cancer Induction><Cancers><Cell Body><Cell Surface Receptors><Cell surface><Cell-Extracellular Matrix><Cells><Chimera Protein><Chimeric Proteins><Chronic><Chronic Periodontitis><Cleaved cell><Closure by Ligation><Clustered Regularly Interspaced Short Palindromic Repeats><Coagulation Factor I><Coagulation Factor One><Coagulation Factor XIII><Collaborations><Combining Site><Complex><Congenic Mice><Congenital afibrinogenemia><Congenital hypofibrinogenemia><Cytotoxin><DNA Endonuclease><DNase I><Data><Defect><Deoxyribonuclease I><Deposit><Deposition><Development><Dropsy><EC 3.4.21.7><ECM><Edema><Endocytosis><Endopeptidase Inhibitors><Enterobacteria phage P1 Cre recombinase><Environment><Enzyme Gene><Enzyme Precursors><Enzymes><Epidermis><Epithelial><Epithelium><Epithelium Part><Esteroproteases><Extracellular Matrix><Extracellular Matrix Degradation><Extracellular Matrix Proteins><FP593><Factor I><Factor I Deficiency><Factor One><Factor XIII><Factor XIII Transamidase><Family><Fibrin><Fibrin Stabilizing Factor><Fibrinase><Fibrinogen><Fibrinogen Deficiency><Fibrinolyses><Fibrinolysin><Fibrinolysis><Fusion Protein><Gene Cluster><Genes><Genetic><Gingiva><Gingival><Glu-Plasmin><Granulocyte Elastase><Green Fluorescent Proteins><Growth Agents><Growth Factor><Growth Substances><Heart><Hereditary factor I deficiency disease><Hereditary hypofibrinogenemia><Homeostasis><Human><Hydrogen Oxide><Hydrops><Image><Imaging Device><Imaging Instrument><Imaging Tool><Impairment><In Vitro><Inflammation><Inflammatory><Integrin Binding><Intoxication><Ion Channel><Ionic Channels><Kidney><Kidney Urinary System><Knock-in><Laki-Lorand Factor><LeTx><Leukocyte Elastase><Ligation><Liver><Location><Lysosomal Elastase><Malignant><Malignant - descriptor><Malignant Neoplasms><Malignant Tumor><Marrow Neutrophil><Marrow monocyte><Mediating><Membrane><Membrane Channels><Mice><Mice Mammals><Modern Man><Molecular><Molecular Interaction><Morphogenesis><Mouse Strains><Murine><Mus><Myelogenous><Myeloid><Myeloid Cells><N-terminal><NH2-terminal><NIAID><NLS Peptide><National Institute of Allergy and Infectious Disease><Natural regeneration><Neutrophil Activation><Neutrophil Elastase><Neutrophilic Granulocyte><Neutrophilic Leukocyte><Nuclear Localization Signal><Nuclear Localization Signal Peptide><Oral><Osteoclastic Bone Loss><Oxygen Radicals><PMN Elastase><Pancreatic DNase><Parodontosis><Pathology><Pathway interactions><Pattern><Peptidase Inhibitors><Peptidases><Peptide Hydrolase Inhibitors><Peptide Hydrolases><Peptide Peptidohydrolase Inhibitors><Periodontal Bone Loss><Periodontal Diseases><Periodontal Resorption><Periodontitis><Physiologic><Physiological><Physiological Homeostasis><Plasmin><Plasminogen><Polymers><Polymorphonuclear Cell><Polymorphonuclear Leukocyte Elastase><Polymorphonuclear Leukocytes><Polymorphonuclear Neutrophils><Pro-Oxidants><Process><Production><Proenzymes><Profibrinolysin><Proprotein Convertases><Protease Antagonists><Protease F><Protease Gene><Protease Inhibitor><Proteases><Protein Cleavage><Proteinase Inhibitors><Proteinases><Proteins><Proteins Growth Factors><Proteolysis><Proteolytic Enzymes><Reactive Oxygen Species><Reactive Site><Receptor Activation><Receptor Protein><Receptor Signaling><Regeneration><Research><Resolution><Serine Endopeptidases><Serine Protease><Serine Protein Hydrolases><Serine Proteinases><Spleen><Spleen Reticuloendothelial System><Squamous Epithelium><Therapeutic Agents><Thrombase><Thrombin><Thymonuclease><Time><Tissues><Toxin><Transgenic Organisms><Water><Wild Type Mouse><Zymogens><alveolar bone><alveolar supporting bone><aminoacid><anthrax lethal factor><anthrax lethal toxin><anthrax toxin><anthrax toxin LF><anthrax toxin receptors><bacteriophage P1 recombinase Cre><bone><cancer microenvironment><capillary><carcinogenesis><cell behavior><cellular behavior><cleaved><cross-link><crosslink><cutaneous barrier><cytokine><developmental><drFP583><ds red protein><dsFP593><edema factor><epidermal barrier><extracellular><fibrinogenase><genetic approach><genetic strategy><hepatic body system><hepatic organ system><human disease><imaging><imaging agent><immunogen><immunopathology><in vivo><inhibitor><inhibitor/antagonist><insight><integrin bound><knockin><lethal factor><lethal toxin><loss of function><malignancy><maxilla alveolar process><membrane structure><monocyte><morphogenetic process><mutant><neoplasm/cancer><neutrophil><new drug treatments><new drugs><new therapeutics><new therapy><next generation therapeutics><novel><novel drug treatments><novel drugs><novel therapeutics><novel therapy><oral cavity epithelium><oral epithelia><oral epithelium><oral tissue><pathway><periodontal disorder><periodontium disease><periodontium disorder><polymerization><prevent><preventing><receptor><receptor binding><receptor bound><red fluorescent protein><regenerate><renal><skin barrier><socket wall><tissue repair><transgenic><tumor><tumor endothelial marker 8><tumor microenvironment><wildtype mouse>