Structural Studies of Optineurin with Related Proteins

• Main Authors: Bai-JiunKuo, 郭佰寯
• Other Authors: Yu-Chih Lo
• Format: Others
• Language: en_US
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/w9p3k2

碩士 === 國立成功大學 === 生物資訊與訊息傳遞研究所 === 102 === Optineurin (OPTN) is a multifunctional protein involved in a lot of signaling pathways, including NFκB activation, autophagy, and innate immune responses, etc. The previous studies have shown that OPTN is a negative regulator which competes with NEMO to bind polyubiqutins in order to down regulating NFκB activation. OPTN is also identified as an autophagy receptor, which can be activated by Tank-binding kinase 1 (TBK1) and subsequently bind with specific cargo and autophagy modifier light chain 3 (LC3) in the selective autophagy response leading to lysosome degradation. Upon viral infection, OPTN will bind to TBK1. However, how OPTN activates or inhibits the activation of the TBK1/IRF3 complex is still unclear. In addition, OPTN mutations are correlated with some diseases such as primary open angle glaucoma, amyotrophic lateral sclerosis (ALS), and Paget disease of bone. According to its crucial role in these pathways and diseases, to figure out how OPTN interacts with its associating proteins in these pathways is the main goal of my thesis. In my study, I designed different lengths of OPTN fragments containing the N-terminal coil-coiled domain, LIR, UBAN for making the complexes with TBK1, LC3, and polyUb, respectively. The protein complexes were expressed in E. coli expression system and the protein complexes were purified by FPLC. I have obtained stable OPTN-polyUb protein complexes from and the crystals from the crystal screening kits for x-ray diffraction. The resolution of best diffraction data set I got so far is 4.5 angstrom I used CCP4i software to performed molecular replacement experiment. However, the resolution is not good enough for me to solve the structure. For the complex of OPTN-LC3, although I have obtained the stable protein complex we could not get the crystals for solving structure. For OPTN-TBK1 I have not obtained any stable protein complex for crystallization. Therefore, my future works will focus on expressing more proteins and getting crystals of higher quality for solving the crystal structures.