Lipids are essential signaling molecules that regulate all aspects of cellular function. Unlike proteins, whose location can be tracked in situ with antibodies or expression of tagged fusion proteins, our ability to track signaling lipids in situ i still primitive and constitutes a major impediment to understanding their role in cell function. Ths is especially true for the signaling lipid ceramide 1- phosphate (C1P), which has been implicated in multiple cellular functions including transmitter exocytosis, inflammation, cell proliferation, protein function regulation, and cytoskeletal dynamics. To overcome this technological challenge we will use deep scanning mutagenesis of the C2 lipid-binding motif, combined with deep sequencing and sequence analysis algorithms, to generate proteins with specific, high-affinity binding to C1P. The resulting proteins will serve as the basis for fluorescent biosensors to track C1P in situ. In addition, the novel protein engineering platform we assemble will be a powerful new strategy for developing new sensors for other lipids.
The signaling lipid ceramide 1-phosphate (C1P) plays a role in many cellular processes relevant to disease. To overcome the lack of approaches to track C1P in cells we will employ deep scanning mutagenesis of the C2 lipid-binding motif, combined with deep sequencing and sequence analysis algorithms, to generate proteins with specific, high-affinity binding to C1P. The resulting proteins will serve as the basis fluorescent biosensors to track this signaling lipid in situ.
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| Bluestein, Blake M; Morrish, Fionnuala; Graham, Daniel J et al. (2016) An unsupervised MVA method to compare specific regions in human breast tumor tissue samples using ToF-SIMS. Analyst 141:1947-57 |
