IDENTIFICATION AND CHARACTERIZATION OF GLUTAREDOXIN1 (GRX1) INTERACTING PROTEINS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Following FasL binding, Fas associates with two specific proteins, Fas associated death domain and caspase-8 to form the death inducing signaling complex. Subsequent activation of caspases -8 & -3, results in cleavage of cellular proteins and apoptosis. Grx1 is an 11 KD peptide that reduces glutathionylated proteins glutathione or back to free sulfhydryls. Recently, we have identified redox modification of Fas itself and subsequent enhancement of cell death. We have also shown a novel regulation of the FasL/Fas pathway by degradation of Grx1 and increase in S-glutathionylation of Fas. The central hypothesis of this project is, upon FasL stimulation Protein S-glutathionylation increases and Grx1 interacts with proteins involved in cell death pathway. In aim 1 we will identify and characterize S-glutathionylated proteins upon FasL stimulation, by using a new technique developed in our lab. In aim 2 we will identify and characterize Grx1 interacting proteins after FasL stimulation by proteomics approach. For this funding period, we have optimized the conditions for immunoprecipitations of Grx1 binding proteins. The preliminary analysis of immunoprecipitated peptides by LC/MS/MS showed that, many cytoskeletal proteins bind to Grx1 after or before stimulation with FasL. After stimulation with FasL the proteins Gelsolin, Vimentin and Fibronectin co-immunoprecipitate with Grx1, but these proteins did not immunoprecipitate along with Grx1 in untreated cells. Further, we are exploring the S-glutathionylation status of these proteins during FasL induced cell death. I am also optimizing and analyzing the in-vitro biotin switch technique where in I reduce the Sglutathionylated proteins using Grx1 and derivatize the newly reduced Cysteins into a stable biotinylated form so that it can be analyzed by LC/MS/MS. In the coming months these optimized techniques will be used to analyze Grx1 interacting proteins and S-glutathionylated proteins in the mouse models of lung fibrosis.