Defensins are tridisulfide peptides implicated in innate immunity against potentially pathogenic microorganisms. Myeloid defensins are packaged in the granules of neutrophils and monocytes, and epithelial defensins are expressed in a wide variety of mucosal tissues. The most recently discovered defensins, termed theta-defensins, are 18-amino acid macrocyclic peptides that are stabilized by three parallel disulfide bonds. Isolated from rhesus monkey leukocytes, theta-defensins are remarkably potent antibiotics that kill bacteria and fungi, and they inactivate HIV-1. Antimicrobial activity is abrogated by opening of the backbone ring. The presence of macrocyclic peptides in animals was not previously known. Moreover, the biosynthesis of theta-defensins is novel, as the cyclic peptide is synthesized from two 9-amino acid segments that are spliced together in a head-to-tail configuration. While the cellular machinery that mediates this post-translational pathway is unknown, we hypothesize that enzymes expressed in theta-defensin-producing cells are responsible for the nonapeptide excision and ligation steps necessary for biosynthesis of the mature cyclic molecule. We propose to characterize the molecular components of the theta-defensin processing pathway by pursuing three specific Aims: 1.
In Specific Aim 1, we will analyze the pro-theta-defensin intermediates produced in myeloid cells, and will determine the subcellular compartments of the molecular intermediates identified. 2.
Specific Aim 2 is to identify pro-theta-defensin converting activities in extracts of theta-defensin-expressing cells. For these studies we will use synthetic and recombinant forms of putative substrates involved in the excision/ligation pathway, and use immunoprecipitation, and chromatographic, electrophoretic, and mass spectroscopic methods for detecting and characterizing the relevant enzymatic activities. 3.
Specific Aim 3 is to characterize proteins that interact with pro-theta-defensins and subsequent intermediates, as these are likely to be convertases or chaperones necessary for the excision/ligation steps involved in theta-defensin biosynthesis. Results obtained from these studies are likely to disclose novel mechanisms that have evolved for splicing and cyclizing proteins in mammalian cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI058129-04
Application #
7238738
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Korpela, Jukka K
Project Start
2004-06-01
Project End
2009-05-31
Budget Start
2007-06-01
Budget End
2008-05-31
Support Year
4
Fiscal Year
2007
Total Cost
$361,494
Indirect Cost
Name
University of California Irvine
Department
Pathology
Type
Schools of Medicine
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
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Schaal, Justin B; Tran, Dat; Tran, Patti et al. (2012) Rhesus macaque theta defensins suppress inflammatory cytokines and enhance survival in mouse models of bacteremic sepsis. PLoS One 7:e51337
Tongaonkar, Prasad; Golji, Amir E; Tran, Patti et al. (2012) High fidelity processing and activation of the human ?-defensin HNP1 precursor by neutrophil elastase and proteinase 3. PLoS One 7:e32469
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Tongaonkar, Prasad; Tran, Patti; Roberts, Kevin et al. (2011) Rhesus macaque ?-defensin isoforms: expression, antimicrobial activities, and demonstration of a prominent role in neutrophil granule microbicidal activities. J Leukoc Biol 89:283-90
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Park, Narae; Yamanaka, Kinrin; Tran, Dat et al. (2009) The cell-penetrating peptide, Pep-1, has activity against intracellular chlamydial growth but not extracellular forms of Chlamydia trachomatis. J Antimicrob Chemother 63:115-23
Tongaonkar, Prasad; Selsted, Michael E (2009) SDF2L1, a component of the endoplasmic reticulum chaperone complex, differentially interacts with {alpha}-, {beta}-, and {theta}-defensin propeptides. J Biol Chem 284:5602-9

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