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Principal Investigator: SAMUEL YANG
Organization: STANFORD UNIVERSITY
Fiscal Year: 2024
Award: $231,600
Funding agency: National Institute of Allergy and Infectious Diseases
PROJECT SUMMARY / ABSTRACT
Neutrophils are the first line of host defense against invading pathogens that trap and kill pathogens through a
process called NETosis. Upon stimulation by the pathogen, neutrophils extrude decondensed chromatin in the
form of DNA fibers that trap invading cells and forms a scaffold to harbor associated proteins and antimicrobial
peptides. Bactericidal activity has been attributed to the action of these associated proteins and peptides through
oxidative stress resulting from the free radicals they generate. DNA however, the largest component of NETs, is
only believed to offer the trapping scaffold despite a suicidal path a neutrophil undergoes to extrude the DNA.
DNA is known to form non-canonical secondary structures like the i-motif, triple helices, and G-quadruplexes
(G4). G4 is formed by the stacking of planar square structures of four guanine bases associated by Hoogsteen
hydrogen binds. These secondary structures are concentrated around transcription start sites in promoter
regions and genome-wide high-resolution sequencing has detected >700,000 G4s in the human genome. G4s
are known to sequester free hemin to protect the cell from hemin toxicity and this complex (G4/H) subsequently
regulates gene expression. In vitro, G4/H has been characterized as a DNAzyme that mimics peroxidases to
decompose to hydrogen peroxide to produce hydroxyl radicals. Hydroxyl radicals are known to be most reactive
and toxic to cells in vivo. While G4/H activity is well understood in vitro and the G4 is known to sequester free
hemin within cells, the existence of G4 structures in the decondensed chromatin of NETs or the peroxidase like
activity of G4/H in vivo is unknown.
We propose to discern the existence of G4/H complexes in NETs using antibodies specific to hemin and G4
using multiple approaches like colocalization of immunofluorescence and ChIP-seq on NETs specific DNA pulled
with myeloperoxidase specific antibodies from whole blood of healthy individuals after IL-8 or bacterial
stimulation and patients with COVID-19. We propose to characterize the enzymatic activity of the G4/H
DNAzyme in NETs and demonstrate the local concentrated effect of free radicals generated on trapped bacteria
and prove that this phenomenon occurs naturally ex vivo. Finally, we propose to exhibit the biological outcomes
from the G4/H DNAzyme generated free radicals by testing bactericidal activity, host cell injury, and
posttranslational modifications of NETs associated proteins giving rise to autoimmune disorders like systemic
lupus erythematosus.
Terms: <3-10C><AMCF-I><Antibodies><Assay><Autoantibodies><Autoimmune Diseases><Bacteria><Binding><Bioassay><Biological><Biological Assay><Blood Neutrophil><Blood Polymorphonuclear Neutrophil><Blood erythrocyte><COVID infected patient><COVID patient><COVID positive patient><COVID-19 infected patient><COVID-19 patient><COVID-19 positive patient><COVID19 patient><COVID19 positive patient><CXCL8><Catalytic DNA><Cell Body><Cell Protection><Cells><Cellular injury><ChIP Sequencing><ChIP-seq><ChIPseq><Chlorohemin><Chromatin><Complex><Cytoprotection><DNA><DNAzymes><Data><Deoxyribonucleic Acid><Deoxyribozymes><Erythrocytes><Erythrocytic><Exhibits><Feedback><Ferriheme Chloride><Ferriprotoporphyrin IX Chloride><Fiber><Free Radicals><G-Quadruplex><G-Quadruplexes DNA><G-Quartet Structures><G-Quartets><G-Tetrads><G4-DNA><GCP1><Gene Expression><Generations><Goals><Guanine><H element><H-bond><H2O2><Hemi-Myeloperoxidase><Hemin><Hemoglobin concentration result><Hemolysis><Histones><Host Defense><Human Genome><Hydrogen><Hydrogen Bonding><Hydrogen Peroxide><Hydroperoxide><Hydroxyl><Hydroxyl Radical><IL-8><IL8><IL8 gene><Immunity><Immunoblotting><Immunochemical Immunologic><Immunofluorescence><Immunofluorescence Immunologic><Immunofluorescence Microscopy><Immunologic><Immunological><Immunologically><Immunologics><Immunology procedure><In Vitro><Individual><Inflammation><Injury><Internet><Invaded><Investigators><K60><Kinetics><Label><Lupus Erythematosus Disseminatus><Marrow Neutrophil><Marrow erythrocyte><Measures><Molecular Interaction><Monitor><Myeloperoxidase><Neutrophilic Granulocyte><Neutrophilic Leukocyte><Outcome><Oxidative Stress><Peptides><Peroxidases><Phenotype><Physiologic><Physiological><Play><Polymorphonuclear Cell><Polymorphonuclear Leukocytes><Polymorphonuclear Neutrophils><Post-Translational Modification Protein/Amino Acid Biochemistry><Post-Translational Modifications><Post-Translational Protein Modification><Post-Translational Protein Processing><Posttranslational Modifications><Posttranslational Protein Processing><Process><Promoter Regions><Promotor Regions><Protein Modification><Proteins><Protohemin><Red Blood Cells><Red Cell><Research Personnel><Researchers><Resolution><Role><SARS-CoV-2 infected patient><SARS-CoV-2 patient><SARS-CoV-2 positive patient><SCYB8><SLE><Sampling><Structure><Surface><Survey Instrument><Surveys><Systemic Lupus Erythematosus><Systemic Lupus Erythematous><Systemic Lupus Erythmatosus><TSG-1><Testing><Toxic effect><Toxicities><Transcription Initiation Site><Transcription Start Site><Visualization><WWW><Western Blotting><Western Immunoblotting><Whole Blood><anti-microbial peptide><autoimmune antibody><autoimmune condition><autoimmune disorder><autoimmunity disease><autoreactive antibody><b-ENAP><bactericidal><bactericide><base><bases><biologic><blood corpuscles><cell damage><cell injury><cellular damage><chromatin immunoprecipitation-sequencing><coronavirus disease 2019 infected patient><coronavirus disease 2019 patient><coronavirus disease 2019 positive patient><coronavirus disease infected patient><coronavirus disease patient><coronavirus disease positive patient><coronavirus disease-19 patient><coronavirus patient><cytoprotective><damage to cells><disseminated lupus erythematosus><erythrolysis><extracellular><genetic promoter element><genetic promoter sequence><genome scale><genome-wide><genomewide><hemoglobin level><human whole genome><immunologic assay><immunologic assay/test><in vivo><injuries><injury to cells><multidisciplinary><neutrophil><pathogen><patient infected with COVID><patient infected with COVID-19><patient infected with SARS-CoV-2><patient infected with coronavirus disease><patient infected with coronavirus disease 2019><patient infected with severe acute respiratory syndrome coronavirus 2><patient with COVID><patient with COVID-19><patient with COVID19><patient with SARS-CoV-2><patient with coronavirus disease><patient with coronavirus disease 2019><patient with severe acute respiratory distress syndrome coronavirus 2><promoter sequence><protein blotting><resolutions><response><scaffold><scaffolding><self reactive antibody><severe acute respiratory syndrome coronavirus 2 infected patient><severe acute respiratory syndrome coronavirus 2 patient><severe acute respiratory syndrome coronavirus 2 positive patient><social role><suicidal><suicidality><systemic lupus erythematosis><triple helix><web><world wide web>