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Title: Characterization of the Acyl-CoA binding domain containing 3 protein 
Author: Li, Zengqiu
Date: 2006
Abstract: The cAMP dependent protein kinase, PKA, is of great importance for cells to transduce extra- and intra-cellular signals. Recent studies have found that signal transduction through PKA is coordinated by a group of scaffold proteins caned A-Kinase Anchoring Proteins. These AKAPs, together with PKA and its substrates, form a dynamic assembly that tightly controls the location and timing of signal transduction events, which in turn regulates various cellular activities including ion channel modulation, cell growth, cell differentiation and cytokinesis. Previous research in Dr. Turner's lab has found that TRPY ion channels recruit PKA through Acyl-CoA Binding Domain containing 3 protein (ACBD3) to transduce physiological stimuli in mast cells and sensory neurons. Other studies have indicated a similar role for ACBD3 during PKA-mediated steroid formation. Hence, we hypothesize that ACBD3 may function as an AKAP recruiting PKA to multiple novel targets, in addition to ion channels such as the TRPVs. The project presented in this paper aimed at production of purified ACBD3 protein, for use as an affinity matrix to purify novel targets of ACBD3. The human ACBD3 cDNA was subcloned into the pTrcHisB vector with a conferred epitope tag comprising six sequential Histidine residues. Then the construct was transformed into the BL21 E.coli strain to express the protein in an inducible manner. Finally, we used Immunobilized Metal Affinity Chromatography (IMAC) technique to purify the fusion protein. With the purified protein, we confirmed the strong PKA binding ability of ACBD3, which is consistent with our hypothesis. We also demonstrated a wide expression profile of ACBD3 in different tissues, suggesting that ACBD3 is likely to function outside the CNS and hence is likely to interact with proteins other than TRP The cAMP dependent protein kinase, PKA, is of great importance for cells to transduce extra- and intra-cellular signals. Recent studies have found that signal transduction through PKA is coordinated by a group of scaffold proteins caned A-Kinase Anchoring Proteins. These AKAPs, together with PKA and its substrates, form a dynamic assembly that tightly controls the location and timing of signal transduction events, which in turn regulates various cellular activities including ion channel modulation, cell growth, cell differentiation and cytokinesis. Previous research in Dr. Turner's lab has found that TRPY ion channels recruit PKA through Acyl-CoA Binding Domain containing 3 protein (ACBD3) to transduce physiological stimuli in mast cells and sensory neurons. Other studies have indicated a similar role for ACBD3 during PKA-mediated steroid formation. Hence, we hypothesize that ACBD3 may function as an AKAP recruiting PKA to multiple novel targets, in addition to ion channels such as the TRPVs. The project presented in this paper aimed at production of purified ACBD3 protein, for use as an affinity matrix to purify novel targets of ACBD3. The human ACBD3 cDNA was subcloned into the pTrcHisB vector with a conferred epitope tag comprising six sequential Histidine residues. Then the construct was transformed into the BL21 E.coli strain to express the protein in an inducible manner. Finally, we used Immunobilized Metal Affinity Chromatography (IMAC) technique to purify the fusion protein. With the purified protein, we confirmed the strong PKA binding ability of ACBD3, which is consistent with our hypothesis. We also demonstrated a wide expression profile of ACBD3 in different tissues, suggesting that ACBD3 is likely to function outside the CNS and hence is likely to interact with proteins other than TRPVs.
Description: Thesis (M.S.)--University of Hawaii at Manoa, 2006. Includes bibliographical references (leaves 53-55). ii, 55 leaves, bound ill. (some col.) 29 cm
URI: http://hdl.handle.net/10125/20721
Rights: All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Keywords: Cooperative binding (Biochemistry)

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