Proteomic Analysis of Neuroendocrine Peptidergic System Disruption Using the AtT20 Pituitary Cell Line as a Model

Abstract

Environmental pollutants may affect the activities of many cellular enzymes. The effect on the proteome of enzymatic inhibitors can be determined using two-dimensional (2D) gel electrophoresis. In neuroendocrine cells, proprotein convertases 1 and 2 (PC1 and PC2) mediate the proteolytic activation of many precursors to peptide hormones and neuropeptides. Enzymatic activities of these calcium-dependent proteinases are readily regulated by chelating agents and by heavy metals ions found in the environment. Such an inhibition could result in a potentially pathological disruption of the peptidergic system. We are interesting in finding out to what extent specific inhibition of these enzymes could affect the proteome of a neuroendocrine cell. To address this question, we used the mouse pituitary AtT20 cell line as a model. We compared the proteomic pattern of control cells to that of cells overexpressing proSAAS, a PC1-specific inhibitor. The comparison was conducted using two-dimensional (2D) gel electrophoresis, mass spectrometric identification of differing proteins and immunoblotting to confirm their identity. The 2D analysis revealed a number of alterations in the proteome of proSAAS-overexpressing cells. Mass spectrometric analysis of tryptic peptides identified two proteins found in more abundance in these cells as proSAAS and Ephrin type A receptor 2.