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This dissertation, Identification of the Protein Interacting Partners of Testis Specific Protein, Y-encoded Like-2 (TSPYL2) by Kit-yan, Yip, 葉潔茵, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Identification of the Protein Interacting Partners of Testis Specific Protein, Y-encoded Like-2 (TSPYL2) Submitted by Yip Kit Yan for the degree of Master of Philosophy at The University of Hong Kong in August, 2007 Abstract Testis-Specific Y-encoded-Like protein 2 (TSPYL2) is a member of the nucleosome assembly protein (NAP) superfamily. All members of this superfamily contain a NAP domain which binds histones. TSPYL2 is expressed in areas with proliferating cells in the fetal and postnatal mouse brain. In mice, cardiac expression of TSPYL2 is switched off within the first week after birth. This coincides with the switch off of cardiomyocyte proliferation. In vitro, TSPYL2 has previously been shown to be expressed in proliferating C2C12 myoblasts. Interestingly, TSPYL2 expression increases on the first day of differentiation but is switched off two days after differentiation. Knockdown of TSPYL2 has previously shown to perturb C2C12 differentiation. Therefore, we hypothesize that TSPYL2 plays a role in proliferation and terminal differentiation of myoblasts. The identification of the interacting partners of TSPYL2 may give insights into its functions and the mechanism involved. Here, I employ both a pull-down approach and a candidate approach to identify the protein-interacting partners of TSPYL2. In the pull-down approach, recombinant proteins containing different domains of mouse TSPYL2 tagged with purification tags were expressed and purified. Initially, NusA-tagged recombinant proteins were used for the pull-down assay. However, the NusA recombinant proteins were found to be unstable. Next, constructs expressing GST-tagged recombinant proteins of the N-terminal domain (GST-N, 1-174 a.a.), N-terminal truncated (GST-NT, 66-677 a.a.), C-terminal domain (GST-C, 411-677 a.a.) and full-length TSPYL2 (GST-FL, 1-677 a.a.) were made. GST-C and GST-NT could not be expressed and this might suggest that bacterial system was not suitable to be used for the expression for these two constructs. GST-FL and GST-N were successfully expressed but only the GST-N could be purified. Heat shock protein Dnaja4 and Neuronal-SCL2 (NSCL2) were pulled down from C2C12 lysates by the purified GST-N. It had previously been shown that NSCL2 was expressed during the first two days after differentiation in C2C12. NSCL2 was chosen for further verification by co-localization in HA-TSPYL2 and myc-NSCL2 co-transfected cells. The two proteins were shown to be partially co-localized. NSCL2 is a neuronal transcription factor which is responsible for DNA-binding. Further experiments are needed to test whether NSCL2 and TSPYL2 co-operate to act as a transcription complex. In the candidate approach, transcription coactivator p300 and cell-cycle activator cyclin B1 were chosen based on two criteria: 1) they interact with other proteins in the NAP superfamily and 2) they have roles in cell cycle progression or the development of heart and brain. Although both candidates were successfully precipitated by their antibodies, they did not co-immunoprecipitate with HA-TSPYL2 under our experimental conditions. This approach may be used in further study for the validation of candidate protein interacting partners identified from pull-down experiments. Whether TSPYL2 interacts with p300 and cyclin B1 remains to be determined. (451 words) DOI: 10.5353/th_b3955784

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