Activated neutrophils (PMNs) play a critical role in sepsis, ischemia-reperfusion injury, and immune complex-mediated diseases. The broad long-term objective of our laboratory is to elucidate the role of Akt in regulating PMN functions. This proposal will test the hypothesis: Hsp27 regulates neutrophil cell survival/ death responses by modulating critical protein-protein interactions within the Akt signalosome by acting as a scaffolding protein.
The specific aims of this proposal are:
Specific Aim 1 - To determine whether Akt associated/dissociated proteins regulate Akt activation and neutrophil apoptosis in the absence of Akt- Hsp27 interaction.
Specific Aim 2 - To determine signaling pathways that underlie Akt-Hsp27 disruption induced neutrophil apoptosis.
Specific Aim 3 - To determine whether induction of neutrophil apoptosis by disruption of Akt-Hsp27 interaction results from changes in phosphorylation and subcellular redistribution of Akt and its candidate proteins.
In specific aim 1 we will generate TAT-fusion proteins to 4 Akt-associating and 4 Akt-dissociating proteins, identified by our proteomic studies and determine effects of transducing these proteins on Akt activation and neutrophil apoptosis. cDNA subcloning, site-directed mutagenesis, protein transductions, cDNA transfections, in vitro kinase assays, and apoptosis assays will be performed to accomplish this aim.
Specific aim 2 experiments will be focused on determining mechanisms that underlie activation of p38 MARK and JNK pathways after disruption of Akt-Hsp27 interaction. We will also investigate the impact of Akt-Hsp27 disruption on the regulation of proteasomal activation. In vitro kinase assays, proteasome activity assays, transduction of relevant TAT-peptides into PMNs will be performed to accomplish this aim.
In specific aim 3, we will focus our work on those Akt-associated or Akt-dissociated proteins shown to regulate Akt activation and neutrophil apoptosis in specific aim 1. We will then determine if these relevant proteins regulate Akt activation and neutrophil apoptosis by undergoing a post-translational modification such as phosphorylation and/or ubiquitination as a consequence of disruption of Akt-Hsp27 interaction. We will also determine if these protein modifications would result in change in localization of these proteins and/or alter their function. TAT-peptide transduction, cell fractionation studies, western blotting, site-directed mutagenesis studies, and confocal microscopy studies will be performed to accomplish this specific aim. These studies will lead us to new targets for disrupting neutrophil-mediated tissue damage in inflammatory diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI075212-02
Application #
7669124
Study Section
Erythrocyte and Leukocyte Biology Study Section (ELB)
Program Officer
Dong, Gang
Project Start
2008-08-08
Project End
2012-07-31
Budget Start
2009-08-01
Budget End
2010-07-31
Support Year
2
Fiscal Year
2009
Total Cost
$331,914
Indirect Cost
Name
University of Louisville
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
057588857
City
Louisville
State
KY
Country
United States
Zip Code
40292
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