Emerging evidence from several laboratories indicates that lipid oxidation products make major contributions to human disease. An important class of lipid oxidation products are electrophiles that react with cellular nucleophiles including glutathione, proteins, and DNA. Although glutathione and DMA modification has been studied for many years, inadequate technology has limited our ability to investigate protein modification and its relation to biological outcome. Technological advances in proteomics and genomics now make it possible to assemble comprehensive profiles of proteins modified by lipid electrophiles as well as the transcriptional changes they induce. Comparison of these dense datasets using systems approaches enables hypotheses to be generated linking protein modification to cellular response. Research in the Program Project over the past two years has generated critical reagents and novel approaches for identifying lipid electrophilemodified proteins and their biological consequences. We are now poised to mount an aggressive campaign to define the chemistry and biology of protein modification by lipid electrophiles and its relationship to inflammation, a major contributor to many human diseases. The role of Project 3 is to identify the most biologically interesting lipid electrophiles emanating from research in Projects 1 and.2 and to define the transcriptional changes that they induce in the human macrophage cell line, THP-1. This information will be compared to the pattern of protein modification by the same agents, which will be determined in Project 4, and hypotheses formulated linking protein modification to biological outcome. These hypotheses will be tested using a combination of approaches such as RNA knockdown, ectopic protein expression, and biochemical assays. This will enable the elucidation of mechanisms by which protein modifications by lipid lectrophiles lead to pro- and anti-inflammatory responses.
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