Optineurin (OPTN) 是一個具有多功能的蛋白，參與在許多訊息傳遞的路徑上，像是NFκB 的活化、自噬作用或者是自體免疫反應。在先前的研究中指出OPTN可藉由與NEMO競爭polyubiquitins來進行NFκB活化的負向調控。OPTN也是自噬作用的受體，當它被TBK1活化後可與將要被自噬的物質及LC3結合形成自噬囊泡，自噬囊泡會再跟溶菌體融合以達成自噬作用。在遭受到病毒感染時OPTN會跟TBK1進行結合，但是OPTN在這條訊息傳遞的路徑上，是促進還是抑制TBK1/IRF3複合體目前仍然尚未釐清。此外，OPTN的突變也跟許多疾病有關，像是原發性開角型青光眼、肌萎縮性脊髓側索硬化症還有佩吉特氏病。OPTN參與了如此多重要的疾病與訊息傳遞路徑，所以我的研究主題希望透過OPTN跟他交互作用的蛋白質之間之結構的研究去探討OPTN在這些訊息傳遞路徑的關係與角色。在我的研究中，我設計了16種不同長度的OPTN，包含了其N端的coil-coiled domain、LIR、UBAN來跟TBK1、LC3還有polyubiquitins結合。利用大腸桿菌的系統去表達蛋白並且利用親和性的純化與高速液態層析儀純化。純化的蛋白質會進行晶體篩選找到適合的長晶條件，經過優化晶體，得到合適的晶體後再以x光來解結構。目前我已經拿到OPTN-polyUb複合體的晶體，也進行x光照射，最好的繞射解析度到達4.5Å。我試著CCP4i軟體的分子置換的方法去解析結構，但是解析度並不夠去解出結構，。另一方面OPTN與OPTN-LC3的蛋白複合體雖然可以有足量的蛋白，但是尚未有晶體可以去解出結構。在OPTN-TBK1沒有辦法拿到得到任何穩定的蛋白質複合物去長晶體。因此，未來會繼續去表達更多蛋白質來得到更高品質的晶體以便於來解析結構。

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.

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