The overall objective of this project is to unravel molecular mechanisms by which reactive oxygen species (ROS) regulate expression of the signal transduction protein G1i2. ROS are implicated in many disease states such as cancer, diabetes, cardiovascular problems, hypertension, aging, and inflammation. To extend preliminary studies showing that G1i2 is up-regulated by ROS through the Nrf2/Keap1 pathway, a pathway that underlies cells'response to oxidative stress, two specific aims are proposed.
In specific aim 1, the objectives are to (a) test the hypothesis that protein-protein interaction between C/EBP1 and one or more of the other transcription factors (Sp1, Nrf2, and NF-kB) that bind at the ROS-responsive region of the G1i2 gene promoter constitute the mechanistic basis for the C/EBP1-induced suppression of G1i2 gene expression, and (b) explore the idea that G1i2 possesses redox-sensing capability. Protein-protein interaction will be monitored by co- immunoprecipitation, co-occupancy on, and/or recruitment (of the transcription factors) to the promoter.
In specific aim 2, the objective is to assess mechanism(s) of the nuclear import of Nrf2, the key transcription factor in the Nrf2/Keap1 pathway. Its nuclear translocation will be monitored by fluorescence imaging of transfected green fluorescence protein (GFP)-tagged Nrf2, and by site-directed mutagenesis to assess the functionality of novel nuclear localization sequences in the protein. Affinity of such sequences to importin alphas that mediate this translocation will be measured by co-immunoprecipitation methods, and kinetics of nuclear import will be measured by fluorescence loss in photo bleaching (FLIP) assays. The work will be done primarily with K562 cells, a hematopoietic cell line, and in some experiments with HepG2 cells which are of liver origin. To elevate ROS levels, the cells will be exposed to the electrophile tert-butylhydroquinone or to H2O2.
. Oxygen radicals, also known as reactive oxygen species (ROS), are products of normal cell metabolism but they are toxic at high levels. They can be induced by drugs, environmental oxidants, and stress-inducing chemicals. ROS and the signal transduction proteins called G proteins are of enormous significance in public health because abnormalities in their function impact many diseases such as diabetes, heart failure, hypertension, atherosclerosis, and cancer. The outcome of the proposed research will contribute to further understanding of these pathologies. Public Health Relevance: The overall objective of this project is to unravel molecular mechanisms by which reactive oxygen species (ROS) regulate expression levels of G1i2, a member of the heterotrimeric G-protein family of signal transduction proteins, and to explore mechanisms of the nuclear import of the transcription factor Nrf2, an upstream event in the ROS-induced upregulation of G1i2. ROS and the signal transduction proteins called G proteins are of enormous significance in public health because abnormalities in their function are implicated in a plethora of diseases and metabolic derangements such as cancer, diabetes, cardiovascular problems vis-`- vis heart failure, hypertension, neurodegenerative disorders, inflammation leading to atherosclerosis, and aging.
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