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.
|Barati, Michelle T; Scherzer, Janice; Wu, Rui et al. (2015) Cytoskeletal rearrangement and Src and PI-3K-dependent Akt activation control GABA(B)R-mediated chemotaxis. Cell Signal 27:1178-1185|
|Jin, Shunying; Merchant, Michael L; Ritzenthaler, Jeffrey D et al. (2015) Baclofen, a GABABR agonist, ameliorates immune-complex mediated acute lung injury by modulating pro-inflammatory mediators. PLoS One 10:e0121637|
|Uriarte, Silvia M; Rane, Madhavi J; Merchant, Michael L et al. (2013) Inhibition of neutrophil exocytosis ameliorates acute lung injury in rats. Shock 39:286-92|
|Uriarte, Silvia M; Rane, Madhavi J; Luerman, Gregory C et al. (2011) Granule exocytosis contributes to priming and activation of the human neutrophil respiratory burst. J Immunol 187:391-400|
|Rane, Madhavi J; Song, Ye; Jin, Shunying et al. (2010) Interplay between Akt and p38 MAPK pathways in the regulation of renal tubular cell apoptosis associated with diabetic nephropathy. Am J Physiol Renal Physiol 298:F49-61|
|Luerman, Gregory C; Uriarte, Silvia M; Rane, Madhavi J et al. (2010) Application of proteomics to neutrophil biology. J Proteomics 73:552-61|
|Merchant, Michael L (2010) Mass spectrometry in chronic kidney disease research. Adv Chronic Kidney Dis 17:455-68|