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Terminal differentiation of skin keratinocytes is a vertically directed multi-step process that is tightly controlled by the sequential expression of a variety of genes. We previously investigated the gene expression profile and found that many of differentiation-related genes expressed in a temporally regulated manner. In this study, we attempted to find the hub-molecules and their intracellular signaling networks during keratinocyte differentiation using in silico analysis of data obtained from previous studies. We used protein-protein interaction prediction software called PSIMAP, and drew a hypothetical signaling network. We chose one candidate hub-molecule SHC1 that were predicted to link EGFR and MAPK signal, and then evaluated the protein-protein interactions experimentally. As predicted, SHC1 bound to the MEK1 in an EGF-regulated manner. Furthermore, SHC1 bound to the MEK1 and p38 MAPK in a keratinocyte differentiation dependent manner. These results demonstrate that in silico protein-protein interaction prediction system can be used to efficiently and cost-effectively select the experimental candidates.

Proteases are involved in various biological phenomena, and their aberrant activity can be an important indicator of disease. Thus, various methods have been developed to analyze the activities of proteases, but their wide application has been hampered because each method has drawbacks. In this report, we propose a new protease assay method based on an engineered autoinhibited protein and enzyme-linked immunoassay (ELISA) in which a protease of interest activates the autoinhibited protein and the signal is amplified via ELISA. Using this concept a sensitive assay method for MMP2 and caspase-3 was developed. The limit of detection for the two proteases was as low as 7 pM for MMP2 and 0.1 pM for caspase-3. The autoinhibited protein is designed modularly, and the new platform is general enough for the development of assay methods for other proteases with minimal modification. [ABSTRACT FROM AUTHOR]