Toxicoproteomic Study of the Disruption of the Neuronal SNARE Complex by the Presence of Lead, Cadmium, Manganese and Nickel
Velda E. Glover, St. John’s University Student
Marc E. Gillespie, Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences,
Abstract: The nervous system is a critical target for heavy metal toxicity. The neuronal SNARE proteins (SNAP 25, VAMP 2 and Syntaxin 1A) are critical for vesicle fusion and their membrane fusing properties are regulated by calcium, making these proteins particularly susceptible to misregulation by heavy metals. In my project a number of metals are used to potentially disrupt the functions of the SNARE complex. Complexes containing neuronal SNARE SNAP25b and gene products from a neuronal human brain cDNA library are screened using a modified yeast two-hybrid system. This methodology is used to detect protein-protein interactions that are disrupted by exogenous toxicants. Four concentrations of each metal were used in addition to controls. The results of three transformations were pooled. Out of one hundred and fifty four transformants, thirty potential interactors were analyzed in the toxicology screen. A colorimetric assay was performed to quantify ?-galatosidase activity. On cadmium plates, five colonies showed disrupted complexes (light blue) as compared to controls (dark blue). For manganese, seven colonies showed disrupted complexes as compared to control. Nickel acted as a microbial and the yeast did not survive the screen at the two highest concentrations. As seen previously in our lab, lead had no toxic effect. Identification of strains containing SNAP25b and novel protein partners disrupted by various heavy metals will lead to a body of data that will be used to discover potential biomarkers of metal toxicity.