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  Global Substrate Profiling of Proteases in Human Neutrophil Extracellular Traps Reveals Consensus Motif Predominantly Contributed by Elastase

O'Donoghue, A. J., Jin, Y., Knudsen, G. M., Perera, N. C., Jenne, D. E., Murphy, J. E., et al. (2013). Global Substrate Profiling of Proteases in Human Neutrophil Extracellular Traps Reveals Consensus Motif Predominantly Contributed by Elastase. PLOS ONE, 8(9): e75141. doi:10.1371/journal.pone.0075141.

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O'Donoghue, Anthony J.1, Author
Jin, Ye1, Author
Knudsen, Giselle M.1, Author
Perera, Natascha C.1, Author
Jenne, Dieter E.2, Author           
Murphy, John E.1, Author
Craik, Charles S.1, Author
Hermiston, Terry W.1, Author
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1external, ou_persistent22              
2Research Group: Enzymes and Inhibitors in Chronic Lung Disease / Jenne, MPI of Neurobiology, Max Planck Society, ou_1950284              

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Free keywords: SERINE PROTEASES; MASS-SPECTROMETRY; SPECIFICITY; INTERLEUKIN-1-BETA; INFLAMMATION; PROTEINASE-3; INHIBITION; ACTIVATION; LIBRARIES; NETOSIS
 Abstract: Neutrophil extracellular traps (NETs) consist of antimicrobial molecules embedded in a web of extracellular DNA. Formation of NETs is considered to be a defense mechanism utilized by neutrophils to ensnare and kill invading pathogens, and has been recently termed NETosis. Neutrophils can be stimulated to undergo NETosis ex vivo, and are predicted to contain high levels of serine proteases, such as neutrophil elastase (NE), cathepsin G (CG) and proteinase 3 (PR3). Serine proteases are important effectors of neutrophil-mediated immunity, which function directly by degrading pathogenic virulent factors and indirectly via proteolytic activation or deactivation of cytokines, chemokines and receptors. In this study, we utilized a diverse and unbiased peptide library to detect and profile protease activity associated with NETs induced by phorbol-12-myristate-13-acetate (PMA). We obtained a "proteolytic signature" from NETs derived from healthy donor neutrophils and used proteomics to assist in the identification of the source of this proteolytic activity. In addition, we profiled each neutrophil serine protease and included the newly identified enzyme, neutrophil serine protease 4 (NSP4). Each enzyme had overlapping yet distinct endopeptidase activities and often cleaved at unique sites within the same peptide substrate. The dominant proteolytic activity in NETs was attributed to NE; however, cleavage sites corresponding to CG and PR3 activity were evident. When NE was immunodepleted, the remaining activity was attributed to CG and to a lesser extent PR3 and NSP4. Our results suggest that blocking NE activity would abrogate the major protease activity associated with NETs. In addition, the newly identified substrate specificity signatures will guide the design of more specific probes and inhibitors that target NET-associated proteases.

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Language(s): eng - English
 Dates: 2013
 Publication Status: Published online
 Pages: 12
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 Rev. Type: Peer
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Title: PLOS ONE
Source Genre: Journal
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Publ. Info: 1160 BATTERY STREET, STE 100, SAN FRANCISCO, CA 94111 USA : PUBLIC LIBRARY SCIENCE
Pages: - Volume / Issue: 8 (9) Sequence Number: e75141 Start / End Page: - Identifier: ISSN: 1932-6203