Immuno-affinity based determination, recombinantproduction and hormonal characterization of tetrahymenathermophila insulin-like protein(s)

The insulin superfamily composed of insulin, insulin-like growth factors (IGF-I and IGF-II), and relaxins regulates glucose metabolism in higher eukaryotes. In literature, the demonstration of the presence of insulin receptors and the induction of glucose uptake by mammalian insulins in lower eukaryotic unicellular Tetrahymena species led to the hypothesis of the presence of genes encoding insulin family members. These target proteins were identified by using the activity-based ammonium sulfate precipitation and immunoprecipitation from T. thermophila, high eukaryotic insulin sequence similarities, the presence of signal peptide, gene size, conserved cysteine motif, and phylogenetic analysis. The first identified T. thermophila insulin-like protein TtINS2 consists of 66 amino acids, and the extracellular signal sequence of 17 amino acids at the N-terminus of this preproprotein was hypothetically determined. The proprotein is 49 amino acids long and contains 7 conserved cysteines. The monomer form of TtINS2-10XHis was produced recombinantly in T. thermophila and secreted out of the cell as 6.5 kDa, like human insulin, forming dimers, tetramers, and hexamers. TtINS2-sfGFP fusion protein was localized to cilia roots/membrane and secretory vesicles according to the fluorescent microscope analysis. Purified TtINS2 protein had hormone activities in T. thermophila that increases cell division and delays cell death, as well as triggered glucose uptake from outside of the cell into the cell. In this thesis study, insulin-like TtINS2 hormone has been characterized for the first time in unicellular organisms (Protista) and thus the existence of insulin hormone family and insulin signaling pathway in low eukaryotes was proved.