OXIDATION AND DEFORMATION IN SICKLE RBC DEHYDRATION

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DESCRIPTION: The objective of this revised renewal application is to improve our understanding of the molecular mechanisms responsible for abnormal dehydration of sickle red cells, arguably the most important cellular defect of these cells and further clarifying the role of iron and oxidation as pathogenetic influences in sickle cell disease. The proposal comprises of two subprojects that are interrelated in that they both are relevant to problems of cation homeostasis and of oxidative pathobiology. The study on the role of oxidation in sickle disease pathophysiology has three specific aims: 1) Identify the reason for increased "autoxidation" of sickle hemoglobin, considering that it derives not from an inherent autoxidative instability but rather from interaction of sickle hemoglobin with transition metals, pathophysiologic oxidants, and membrane phospholipid; 2) Define the nature of iron decompartmentalization in sickle red cells by assessing: cytoplasmic free transition metal content and its relationship to membrane-free iron, as well as equilibrium binding of iron as influenced by cytosolic physiologic chelators; the role of metal/hemoglobin/membrane interaction in membrane iron deposition; and iron compartments as a function of genotype; 3) Test predictions about the biochemical consequences of such oxidative processes including the validating of hypotheses: that unstable hemoglobinopathy membranes are sickle- like; and that removal of red cell membrane iron using an iron chelator should effect predictable improvements, an approach that will test a potential therapeutic option. The study on the role of membrane deformation-induced cation leak from red cells has three specific aims: 1) Define mechanisms explaining the unique features of the leak resulting from hypotonic deformation. The leading hypothesis to be tested is that macromolecular crowding exerts a major influence on membrane's response to deformation; 2) Establish the relationship of this model to authentic sickling by testing the prediction that the qualitative character of the sickling-induced leak will depend on the degree of deformation; detailing its comparative phenomenology; and determining its relationship to membrane lipid hydroperoxides; and 3) Approach the question as to whether red cell mechanosensitivity occurs by design or accident by examining the deformation response of selected red cells with known leak pathway deficiencies; and by using zinc treated red cells to examine the hypothesis that a permissive mechanosensitive leak structure is formed by clustering of Band 3 protein. It is anticipated that successful accomplishment of these objectives will contribute to a significant improvement in our understanding of the molecular basis for sickle red cell membrane pathobiology and cell dehydration and could also lead to novel therapeutic approaches to sickle cell disease.