Despite the fact that lead poisoning affects almost one in twenty young children in the US, the mechanism of lead poisoning is not understood at the molecular level. Recent advances in neurobiology and human genetics have pinpointed two proteins that are likely targets for lead in vivo: synaptotagmin (Syt) and delta-aminolevulinic acid dehydratase (ALAD). The goals of the proposed research are to elucidate the mechanisms by which lead inactivates these critical metalloproteins. Specifically, the aims of this study are: 1: To determine whether Pb+2 blocks Ca+2 from binding Syt. 2: To determine whether calcium mediates the activity of Syt by causing a change in the association state of the C2 domains and whether Pb+2 interferes with this process. 3: To determine how lead interferes with formation of the synaptotagmin-syntaxin complex by testing whether Pb+2 destabilizes syntaxin and by determining the metal-dependence of the association between Syt and Syn. 4: To determine whether the metal-binding properties of ALAD1 and ALAD2 account for the phenotypes observed for the two isoforms. By studying lead's interactions and effects on each of these proteins we seek to gain a broad understanding of the mechanisms of lead toxicity. Furthermore, the studies proposed herein will provide the foundation for future studies by providing methodologies for studying lead-protein interactions that can then be extended to new protein targets as they are identified.
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