IDENTIFICATION AND CHARACTERIZATION OF GLUTAREDOXIN1 (GRX1) INTERACTING PROTEINS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 Fas与FasL结合后，与Fas相关的死亡结构域和caspase-8两种特定蛋白结合，形成死亡诱导信号复合体。随后激活caspase-8和-3，导致细胞蛋白的裂解和细胞凋亡。Grx1是一种11kD的多肽，可将谷胱甘肽或还原型谷胱甘肽还原为游离的巯基。最近，我们发现了Fas自身的氧化还原修饰和随后的细胞死亡增强。我们还发现了一种通过降解Grx1和增加Fas的S谷胱甘肽基化来调节FasL/Fas通路的新途径。该项目的中心假设是，在FasL刺激下，蛋白S谷胱甘肽基化增加，Grx1与参与细胞死亡途径的蛋白相互作用。在目标1中，我们将利用我们实验室开发的一项新技术，鉴定和表征FasL刺激下的S谷胱甘肽基化蛋白。在目标2中，我们将用蛋白质组学的方法鉴定和鉴定FasL刺激后Grx1相互作用的蛋白。 在这一资助期间，我们优化了Grx1结合蛋白的免疫沉淀条件。免疫沉淀肽的LC/MS/MS初步分析表明，在FasL刺激前后，许多细胞骨架蛋白与Grx1结合。经FasL刺激后，明胶蛋白、波形蛋白和纤维连接蛋白与Grx1免疫共沉淀，但这些蛋白在未处理的细胞中不与Grx1一起免疫共沉淀。此外，我们正在探索这些蛋白在FasL诱导的细胞死亡过程中的S谷胱甘肽基化状态。我还在优化和分析体外生物素切换技术，在该技术中，我使用Grx1还原谷胱甘肽结合蛋白，并将新还原的半胱氨酸衍生为稳定的生物素化形式，以便可以用LC/MS/MS进行分析。在接下来的几个月里，这些优化的技术将被用于分析Grx1相互作用蛋白和S-谷胱甘肽基化蛋白在小鼠肺纤维化模型中的作用。

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.