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Autophagy. 2015 Apr 3;11(4):685-700. doi: 10.4161/auto.36098.

Mutations in the ubiquitin-binding domain of OPTN/optineurin interfere with autophagy-mediated degradation of misfolded proteins by a dominant-negative mechanism.

Author information

1
a Taiwan International Graduate Program in Molecular Medicine; National Yang-Ming University and Academia Sinica ; Taipei , Taiwan.

Abstract

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.

KEYWORDS:

ALS, amyotrophic lateral sclerosis; Ab, antibody; BafA1, bafilomycin A1; CCD, coiled-coil domain; Ef, FRET efficiency; FT, filter-trap assay; HD, Huntington disease; IBs, inclusion bodies; IP, immunoprecipitation; K48, lysine 48; K63, lysine 63; LIR, LC3-interacting region; MYO6, myosin VI; OPTN, optineurin; PBS, phosphate-buffered saline; PFA, paraformaldehyde; TARDBP/TDP-43; TARDBP/TDP-43, TAR DNA-binding protein; TBK1, TANK-binding kinase 1; TUBA, alpha tubulin; UPS, ubiquitin-proteasome system; Ub, ubiquitin B/C/D; UbBD, ubiquitin-binding domain; WB, western blot; WT, wild type; autophagy; dominant-negative; huntingtin; mHTT, mutant huntingtin; optineurin

PMID:
25484089
PMCID:
PMC4502753
DOI:
10.4161/auto.36098
[Indexed for MEDLINE]
Free PMC Article

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