Shen Wen-Chuan, Li Huei-Ying, Chen Guang-Chao, Chern Yijuang, Tu Pang-Hsien
a Taiwan International Graduate Program in Molecular Medicine; National Yang-Ming University and Academia Sinica ; Taipei , Taiwan.
Autophagy. 2015 Apr 3;11(4):685-700. doi: 10.4161/auto.36098.
OPTN (optineurin) is an autophagy receptor and mutations in the OPTN gene result in familial glaucoma (E50K) and amyotrophic lateral sclerosis (ALS) (E478G). However, the mechanisms through which mutant OPTN leads to human diseases remain to be characterized. Here, we demonstrated that OPTN colocalized with inclusion bodies (IBs) formed by mutant HTT/huntingtin protein (mHTT) in R6/2 transgenic mice and IBs formed by 81QNmHTT (nuclear form), 109QmHTT (cytoplasmic form) or the truncated form of TARDBP/TDP-43 (TARDBP(ND251)) in Neuro2A cells. This colocalization required the ubiquitin (Ub)-binding domain (UbBD, amino acids 424 to 511) of OPTN. Overexpression of wild-type (WT) OPTN decreased IBs through K63-linked polyubiquitin-mediated autophagy. E50K or 210 to 410Δ (with amino acids 210 to 410 deleted) whose mutation or deletion was outside the UbBD decreased the IBs formed by 109QmHTT or TARDBP(ND251), as was the case with WT OPTN. In contrast, UbBD mutants, including E478G, D474N, UbBDΔ, 411 to 520Δ and 210 to 520Δ, increased accumulation of IBs. UbBD mutants (E478G, UbBDΔ) retained a substantial ability to interact with WT OPTN, and were found to colocalize with polyubiquitinated IBs, which might occur indirectly through their WT partner in a WT-mutant complex. They decreased autophagic flux evidenced by alteration in LC3 level and turnover and in the number of LC3-positive puncta under stresses like starvation or formation of IBs. UbBD mutants exhibited a weakened interaction with MYO6 (myosin VI) and TOM1 (target of myb1 homolog [chicken]), important for autophagosome maturation, in cells or sorted 109QmHtt IBs. Taken together, our data indicated that UbBD mutants acted as dominant-negative traps through the formation of WT-mutant hybrid complexes to compromise the maturation of autophagosomes, which in turn interfered with OPTN-mediated autophagy and clearance of IBs.
视神经病相关蛋白（OPTN）是一种自噬受体，OPTN基因突变会导致家族性青光眼（E50K）和肌萎缩侧索硬化症（ALS）（E478G）。然而，突变型OPTN导致人类疾病的机制仍有待阐明。在此，我们证明在R6/2转基因小鼠中，OPTN与突变型亨廷顿蛋白（mHTT）形成的包涵体（IBs）共定位，在Neuro2A细胞中，OPTN与81QNmHTT（核形式）、109QmHTT（胞质形式）或TARDBP/TDP-43截短形式（TARDBP(ND251)）形成的IBs共定位。这种共定位需要OPTN的泛素（Ub）结合结构域（UbBD，氨基酸424至511）。野生型（WT）OPTN的过表达通过K63连接的多聚泛素介导的自噬减少了IBs。E50K或210至410Δ（缺失氨基酸210至410），其突变或缺失位于UbBD之外，与WT OPTN一样，减少了由109QmHTT或TARDBP(ND251)形成的IBs。相反，包括E478G、D474N、UbBDΔ、411至520Δ和210至520Δ在内的UbBD突变体增加了IBs的积累。UbBD突变体（E478G、UbBDΔ）保留了与WT OPTN相互作用的显著能力，并且被发现与多聚泛素化的IBs共定位，这可能是通过它们在WT-突变体复合物中的WT伙伴间接发生的。它们降低了自噬通量，这在饥饿或形成IBs等应激条件下，通过LC3水平和周转率以及LC3阳性斑点数量的改变得以证明。UbBD突变体在细胞或分选的109QmHtt IBs中与对自噬体成熟很重要的肌球蛋白VI（MYO6）和myb1同源物（鸡）的靶标（TOM1）的相互作用减弱。综上所述，我们的数据表明，UbBD突变体通过形成WT-突变体杂合复合物作为显性负性陷阱，损害自噬体的成熟，进而干扰OPTN介导的自噬和IBs的清除。
