Optineurin的结构、功能与泛素化修饰的调控机制

Optineurin (OPTN)encodes a 67 KD protein that features mutiple domains that each specifies a set of interactions. OPTN is known to play critical roles in regulating NF-κB signaling, cell polarity,vesicle transportation, Golgi assembly, autophagy etc. Recently, mutations in OPTN were shown to be strongly associated with various types of Glaucoma and Amyotrophic lateral Sclerosis (ALS) with mechanism yet to be understood. Previous studies also revealed that wildtype OPTN is ubiquitylated in vivo and degraded through ubiquitin-proteasome pathway, while disease-associated OPTN is degraded via autophagy pathway. E3 Ub ligases specifically bind to and ubiquitylate OPTN are yet unknown. ..In this application, we aim to understand the molecular basis for the pathophysiological roles of OPTN and its disease-related mutants that OPTN plays in health and diseases, using structure and functional biology approaches. Specifically, we set out to obtain 3-D structure of OPTN from human and other origins, while biochemical and cell-based proteomic analysis are carried out to identify OPTN protein complexes, with an emphasis on an OPTN-interacting E3 Ub ligase， E3X. In the meantime, we have also contructed OPTN conditional knockout mice and the cell lines, which would play a solid foundation for further functional study of the multi-tasking protein, OPTN.

Optineurin(OPTN)是一个多结构域，分子量为67KD的蛋白。OPTN参与调节NF-κB信号通路、细胞极性、囊泡运输、高尔基体组装、细胞自噬和细胞迁移等。研究表明OPTN的突变与人类青光眼、脊髓侧索硬化症等有关，但其作用机制并不清楚。研究表明OPTN本身的稳定性受泛素－蛋白酶体途径调节，而疾病相关的突变则改变其降解途径，目前使OPTN 泛素化的泛素链接酶却仍然未知。我们计划一方面通过与晶体学家合作获得OPTN高分辨率的结构，同时通过蛋白质组学、生物化学及分子生物学的方法鉴定与OPTN的结合蛋白特别是泛素链接酶，研究泛素化对其稳定性和生理功能的调节机制；同时建立OPTN条件型敲除小鼠的动物和细胞模型研究OPTN的生理功能,并且通过研究其疾病相关突变体的病理意义。

自噬(Autophagy)途径是特异性受体蛋白介导的胞内大分子通过主动性包被、吞噬后在溶酶体中选择性降解的过程，而自噬蛋白质受体如OPTN、p62/SQSTM1在生理、病理状态中的作用机制尚不明确。我们通过 OPTN 泛素化修饰E3连接酶的鉴定、蛋白结构的分析、基于肿瘤细胞和动物模型的作用机制研究发现E3连接酶HACE1特异性介导OPTN 蛋白Lys193位点K48为主的多泛素化修饰，多泛素化的OPTN结合自噬受体分子p62形成受体蛋白复合物进而激活细胞自噬，而OPTN本身被多泛素化后通过溶酶体依赖的自噬途径被降解；临床样本和裸鼠成瘤模型表明HACE1-OPTN-p62复合物通过调节肺癌肿瘤细胞的自噬发挥其抑制肿瘤的功能。本项研究发现HACE1-OPTN调控p62介导的自噬这一生理过程的作用机制，揭示了通过自噬调控肿瘤发生发展一个作用机理，为以自噬为靶向的肿瘤药物开发提供一个新的策略。