| Summary: | 碩士 === 國立中正大學 === 生物醫學研究所 === 99 === In our body, pancreas plays a critical role in maintaining endocrine, exocrine, and metabolism homeostasis. Recent studies have identified a network of transcription factors, including Pdx1, Ptf1a, and Ngn3, regulating the development of pancreas. Particularly, Neurogenin-3 (Ngn3) has been shown to be required for the development of all endocrine cell lineages of the pancreas. Besides, Ngn3-deficient mice fail to generate any endocrine progenitor cells.
However the molecular mechanisms controlling the specification of pancreatic endocrine precursors remained unknown. Previously, our laboratory has utilized the yeast two-hybrid assay to find the proteins which can interact with Ngn3. After screening, we have identified several genes, including CCAR1, which can interact with Ngn3. Hence, we want to explore the role of CCAR1 for Ngn3. Because of the interaction between Ngn3 and CCAR1 has confirmed. But the CCAR1 from screening of yeast two-hybrid assay is amino acid 538~723, △CCAR1, not the CCAR1 full lentgh. Although the △CCAR1 had 186 amino acid but it included two different domain, SAP-domain and EDK-rich domain. Then, in my study, I want to prove which domain Ngn3 bound to. I have confirmed the protein interactions between CCAR1-EDK-rich domain and Ngn3 using pull-down assay. And about the functional assay of CCAR1, I used report assay to find the role of CCAR1 in pancreatic development. From our data of Luciferase reporter assay, the effect of Ngn3 would be down-regulated when the expression of CCAR1 was knocked down. And in the other hand, when we increased the expression of CCAR1, the activity of Luciferase would be raised by Ngn3 was up-regulated. Hence, it is also likely that CCAR1 may mediate pancreatic endocrine differentiation together with Ngn3.
Besides, we speculated that CCAR1 is direct targets of Ngn1 and Ngn2. We confirmed the hypothesis by pull-down assay. But the result of pull-down assay just explained the direct interaction between the purified protein of Ngn1, Ngn2, and CCAR1. In the future, we should use co-immunoprecipitation assay to prove the interaction. And then, we could also found the effect of CCAR1 in neuron development.
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