Rapid Quantification of Nuclear Citrullination in Human Neutrophils

PROJECT SUMMARYChromatin decondensation is controlled in part by the PADdependent conversion of arginine to citrulline on histonesi ehistone citrullinationPADis highly expressed in neutrophils and is the only member of the peptidylarginine deiminasePADfamily expressed in the nucleusHypercitrullination by PADis pathophysiological and associated with many diseasese gautoimmune diseaseinflammationand cancerPADactivation in neutrophils initiates leukotoxic hypercitrullinationLTHa cell death program distinct from apoptosisnecrosisor traditional NETosiswherein chromatin is hypercitrullinateddecondensedand extruded from the cell in NET like structuresOf noteLTH NETsneutrophil extracellular trapsare distinct from NADPH oxidase dependent NETsi etraditional NETosisan antibiotic form of neutrophil cell deathwhich do not contain citrullinated histonesThe release of citrullinated chromatin into the blood is thought to contribute directly to PADrelated pathologiesThusPADis a valuable drug target and nucleosome citrullination is a promising blood accessible biomarkerThere are no quantitative assays capable of reliably quantifying PADdependent nucleosome citrullination in human neutrophils or bloodCurrent PADELISAs are low throughput and use citrullinated histonesCitHisas calibration standardsCitHis are problematic for quantification for two major reasonsFirstthey fail to provide accurate quantification of nucleosomes at physiological levels observed in cellssecondfree histonesnot assembled into a nucleosomereadily aggregate in plasmathereby limiting their use in bloodbased samplesHerewe propose to develop the first assayCitNucTMto accurately quantify histone citrullination for next generation drug discovery and biomarker developmentOur innovative assay uses chemically defined recombinant citrullinated designer nucleosomesCitHdNucsas quantification standards for reliable assay calibrationUnlike histonescitrullinated nucleosomes provide useful standards at low concentration and do not aggregate in heterogeneous biological solutionsi eplasmaMoreoversince CitHdNucs resemble chromatin in vivosamples require minimal processing enabling development of the first highthroughputHTcell based assays for PADinhibitor developmentIn Aimwe will develop the CitNuc assayon AlphaLISA platformThis high throughput compatible assay will be useful for drug development and basic research applicationsIn Aimwe will develop the pre clinical application of our CitNuc assay to quantify citrullinated nucleosomes levels in plasmaTo validate this assaywe will compare citrullinated nucleosome levels in patients with rheumatoid artheritisRAa disease characterized by high levels of citrullinated nucleosomes in bloodIn Phase IIwe will scale up commercial manufacturing of CitHdNucsdevelop novel methods for increased assay throughput for inhibitor screeningand expand our pre clinical studies to develop the utility of our assay for disease monitoring in RA patients!PROJECT NARRATIVEPeptidyl Arginine DeiminasesPADis highly expressed in neutrophils and is the only member of the PAD family expressed in the nucleusPADdependent conversion of arginine to citrulline on nucleosomes is pathophysiological and associated with many diseasesincluding inflammation and autoimmune diseaseThusPADis a valuable drug target and nucleosome citrullination is a promising blood accessible biomarkerHowevercurrent cell based assays used to measure nucleosome citrullination are not quantitativeTo meet this needEpiCypher will develop CitNucTMa novel assay platform that utilizes citrullinated designer nucleosomes for sensitive and accurate quantification of PADdependent nuclear citrullination!