The central objective of this research is to use structure-based mimetics to elucidate the topology of receptor recognition surfaces in interleukin 5 (IL5) and to advance mechanistic understanding of IL5 specifically and 4-helix bundle growth factor proteins generally. IL-5 is the major hematopoietin which stimulates the proliferation, migration and activation of eosinophils and has been implicated in the pathogenesis of diseases such as asthma. IL5 is a homodimeric protein, dominated by two 4-helix bundle conformational domains. It functions through a cell- surface receptor that contains an alpha chain that binds IL5 selectively and a more 'public' Beta chain for signal transduction. Available data for IL5 provide an important opportunity to use recognition site mimetics to decipher basic structural principles about how the 4-helix bundle framework in IL5 displays receptor binding epitopes for alpha and Beta chains. This project has several specific aims: (1) based on the topology of receptor recognition residues in hIL5, design chimeric cytokine hybrids of IL5, using both a 2-bundle framework (IL5 type) and 1-bundle framework (GM-CSF type), and compare their binding and structural properties, including testing the ability of IL5 to fold and function as a one 4- helix bundle monomer vs the naturally occuring 2-bundle dimer; (2) design and characterize de novo mimetics of IL5 by transplanting key helical and loop surfaces, as defined by topological mapping and hybrids, into structure-selected scaffolds including the recently developed coiled coil stem loop miniprotein; (3) design contact residue topology mimetics from CDRs of neutralizing anti-[IL5 receptor] monoclonal antibodies; (4) perform followup mutagenesis of hIL5, using a newly designed, biologically active single chain construct of hIL5 (scIL5), in order to verify the importance of residues or residue clusters determined to be important for receptor recognition by cytokine hybrids and de novo mimetics and also to construct advanced receptor binding site topology maps. Long term, this project will lead to a fundamental understanding of recognition epitopes within the structural architecture of IL5. It will yield models for how variations within the basic 4-helix bundle architecture, common to many growth factor proteins, trigger selective receptor recognition. And, it will provide mimetic strategies useful to design 4-helix bundle cytokine antagonists.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI040462-01
Application #
2005042
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Project Start
1996-12-01
Project End
2000-11-30
Budget Start
1996-12-01
Budget End
1997-11-30
Support Year
1
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Ishino, Tetsuya; Economou, Nicoleta J; McFadden, Karyn et al. (2011) A protein engineering approach differentiates the functional importance of carbohydrate moieties of interleukin-5 receptor ýý. Biochemistry 50:7546-56
Zaks-Zilberman, Meirav; Harrington, Adrian E; Ishino, Tetsuya et al. (2008) Interleukin-5 receptor subunit oligomerization and rearrangement revealed by fluorescence resonance energy transfer imaging. J Biol Chem 283:13398-406
Ishino, Tetsuya; Harrington, Adrian E; Zaks-Zilberman, Meirav et al. (2008) Slow-dissociation effect of common signaling subunit beta c on IL5 and GM-CSF receptor assembly. Cytokine 42:179-90
Bhattacharya, Madhushree; Pillalamari, Udaya; Sarkhel, Sanjay et al. (2007) Recruitment pharmacophore for interleukin 5 receptor alpha antagonism. Biopolymers 88:83-93
Ishino, Tetsuya; Pillalamarri, Udaya; Panarello, Dominick et al. (2006) Asymmetric usage of antagonist charged residues drives interleukin-5 receptor recruitment but is insufficient for receptor activation. Biochemistry 45:1106-15
Ishino, Tetsuya; Robertson, Noreen; Chaiken, Irwin (2005) Cytokine recognition by human interleukin 5 receptor. Vitam Horm 71:321-44
Ishino, Tetsuya; Urbina, Cecilia; Bhattacharya, Madhushree et al. (2005) Receptor epitope usage by an interleukin-5 mimetic peptide. J Biol Chem 280:22951-61
Ishino, Tetsuya; Pasut, Gianfranco; Scibek, Jeffery et al. (2004) Kinetic interaction analysis of human interleukin 5 receptor alpha mutants reveals a unique binding topology and charge distribution for cytokine recognition. J Biol Chem 279:9547-56
Ruchala, Piotr; Varadi, Gyorgyi; Ishino, Tetsuya et al. (2004) Cyclic peptide interleukin 5 antagonists mimic CD turn recognition epitope for receptor alpha. Biopolymers 73:556-68
Li, Chuanzhao; Plugariu, Carmela G; Bajgier, Joanna et al. (2002) Coiled coil miniprotein randomization on phage leads to charge pattern mimicry of the receptor recognition determinant of interleukin 5. J Mol Recognit 15:33-43

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