We propose to characterize that subset of the proteome possessing a particular type of post-translational modification protein carbonyls that has been associated with diverse pathologies including inflammatory conditions, neurodegenerative diseases, aging and anemia. Our goal is to develop improved reagents and techniques for study of the role of protein carbonylation in cellular physiology in both normal and pathologic states. Improved technical ability will facilitate investigation of a number of basic questions regarding protein oxidation: What is the hierarchy of protein oxidation -- are there specific proteins that are carbonylated first, which serve as a buffer against oxidative insult? Carbonylated proteins are subject to degradation in the proteasome --does degradation of `carbonyl sensor' proteins activate cellular responses to oxidative damage? Are there distinct signatures or patterns of protein oxidation that are disease specific? If so, can these patterns provide useful diagnostic or prognostic information? In this proposal we will use an established mouse model of Sideroblastic Anemia (SA), in which protein carbonyls are significantly elevated, as substrate for the development and validation of novel techniques for the enrichment, quantification and identification of proteins possessing this type of oxidative lesion. We will then use these methods in the analysis of purified sideroblasts (iron loaded cells characteristic of this disorder) isolated from clinical specimens of patients with SA. We present a novel method for purification of these iron loaded cells from patient samples that will facilitate these studies. Analysis of oxidized proteins will employ affinity purification, differential gel analysis (DIGE) and mass spectrometry to define the oxidized proteome in both the mouse model and clinical specimens. ? ? ?
| Nagababu, E; Mohanty, J G; Friedman, J S et al. (2013) Role of peroxiredoxin-2 in protecting RBCs from hydrogen peroxide-induced oxidative stress. Free Radic Res 47:164-71 |
| Xu, Xiuling; Martin, Florent; Friedman, Jeffrey S (2010) The familial Parkinson's disease gene DJ-1 (PARK7) is expressed in red cells and plays a role in protection against oxidative damage. Blood Cells Mol Dis 45:227-32 |
| Martin, Florent M; Prchal, Josef; Nieva, Jorge et al. (2008) Purification and characterization of sideroblasts from patients with acquired and hereditary sideroblastic anaemia. Br J Haematol 143:446-50 |
