Cyclophilin A (CypA/Ppia) is an 18kD, 8-stranded beta-barrel, with a solvent-exposed hydrophobic pocket.The immunosuppressive drug cyclosporine grips the pocket and with CypA creates a composite surface that binds and inhibits calcineurin. The pocket cradles proline-containing peptides, including an exposed proline on HIV-1 Capsid, and catalyzes cis-trans interconversion of the peptidyl-prolyl bond. To identify the native function of CypA we generated mice lacking Ppia. Ppia1'mice develop allergic pathology with tissue infiltration by eosinophils and mast cells, and increased CD4+ T-helper type II (Th2) cytokine production. We hypothesized that the pathology resulted from increased activity of Itk, a tyrosine kinase upstream of PLC gamma (and therefore of calcineurin) that specifically promotes Th2 function. CypA bound Itk via the PPIase active site. Mutation of a conformationally heterogeneous proline in Itk (P287G) disrupted CypA binding and conferred Th2 hyperactivity on wild-type CD4+ T cells. Thus, CypA regulates CD4+ T cells signal transduction in the absence of cyclosporine via a regulatory proline residue in Itk.
Aim 1 : Ppia4'mice exhibit strain-dependent lethality. A dominant suppressor of lethality in 129/SvEv mice will be mapped and cloned.
Aim 2 : The functional significance of CypA peptidyl-prolyl isomerase activity for inhibition of Itk and Th2 cytokine expression will be tested using a panel of rationally-designed CypA mutants, as well as mutants screened in a yeast strain that is dependent on CypA PPIase activity.
Aim 3 : Ppia*'* memory CD4+ Th2 cells secrete enormous quantities of IL2. We will determine if this phenotype also results from effects of CypA on Itk and we will elucidate the post-transcriptional mechanism underlying it. These studies will provide the first detailed assessment of the biological function of CypA, shed light on a novel mechanism of protein kinase regulation characterized by a conformational switch in Itk, and advance basic knowledge of protein-folding, T cell signaling, cytokine expression, and CD4+ T cell differentiation. New information concerning these basic biological processes will help understand the etiology of such diseases as asthma and AIDS, and, therefore, is critical for the development of much-needed medical therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI059159-06
Application #
7758377
Study Section
Cellular and Molecular Immunology - B Study Section (CMIB)
Program Officer
Mallia, Conrad M
Project Start
2006-01-15
Project End
2012-08-31
Budget Start
2011-01-01
Budget End
2012-08-31
Support Year
6
Fiscal Year
2011
Total Cost
$247,210
Indirect Cost
Name
University of Geneva
Department
Type
DUNS #
481076537
City
Geneva
State
Country
Switzerland
Zip Code
CH-12-11
Pizzato, Massimo; McCauley, Sean Matthew; Neagu, Martha R et al. (2015) Lv4 Is a Capsid-Specific Antiviral Activity in Human Blood Cells That Restricts Viruses of the SIVMAC/SIVSM/HIV-2 Lineage Prior to Integration. PLoS Pathog 11:e1005050
De Iaco, Alberto; Luban, Jeremy (2014) Cyclophilin A promotes HIV-1 reverse transcription but its effect on transduction correlates best with its effect on nuclear entry of viral cDNA. Retrovirology 11:11
Bartels, Hanni; Luban, Jeremy (2014) Gammaretroviral pol sequences act in cis to direct polysome loading and NXF1/NXT-dependent protein production by gag-encoded RNA. Retrovirology 11:73
Yang, Yang; Luban, Jeremy; Diaz-Griffero, Felipe (2014) The fate of HIV-1 capsid: a biochemical assay for HIV-1 uncoating. Methods Mol Biol 1087:29-36
De Iaco, Alberto; Santoni, Federico; Vannier, Anne et al. (2013) TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm. Retrovirology 10:20
Miyamoto, Tadashi; Nakayama, Emi E; Yokoyama, Masaru et al. (2012) The carboxyl-terminus of human immunodeficiency virus type 2 circulating recombinant form 01_AB capsid protein affects sensitivity to human TRIM5α. PLoS One 7:e47757
Luban, Jeremy (2012) Innate immune sensing of HIV-1 by dendritic cells. Cell Host Microbe 12:408-18
Luban, Jeremy (2012) TRIM5 and the Regulation of HIV-1 Infectivity. Mol Biol Int 2012:426840
Grütter, Markus G; Luban, Jeremy (2012) TRIM5 structure, HIV-1 capsid recognition, and innate immune signaling. Curr Opin Virol 2:142-50
Singh, Ravesh; Gaiha, Gaurav; Werner, Lise et al. (2011) Association of TRIM22 with the type 1 interferon response and viral control during primary HIV-1 infection. J Virol 85:208-16

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