Structural Studies of Optineurin with Related Proteins

  • 郭 佰寯

Student thesis: Master's Thesis

Abstract

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
Date of Award2014 Aug 22
Original languageEnglish
SupervisorYu-Chih Lo (Supervisor)

Cite this

Structural Studies of Optineurin with Related Proteins
佰寯, 郭. (Author). 2014 Aug 22

Student thesis: Master's Thesis