Demirsoy S, Martin S, Motamedi S, van Veen S, Holemans T, Van den Haute C, Jordanova A, Baekelandt V, Vangheluwe P, Agostinis P
Laboratory for Cell Death Research and Therapy, Department of Cellular and Molecular Medicine, University of Leuven (KU Leuven).
Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, University of Leuven (KU Leuven), Campus Gasthuisberg, O&N1, Herestraat 49, Box 802, B-3000 Leuven, Belgium.
Hum Mol Genet. 2017 May 1;26(9):1656-1669. doi: 10.1093/hmg/ddx070.
ATP13A2 (also called PARK9), is a transmembrane endo-/lysosomal-associated P5 type transport ATPase. Loss-of-function mutations in ATP13A2 result in the Kufor-Rakeb Syndrome (KRS), a form of autosomal Parkinson's disease (PD). In spite of a growing interest in ATP13A2, very little is known about its physiological role in stressed cells. Recent studies suggest that the N-terminal domain of ATP13A2 may hold key regulatory functions, but their nature remains incompletely understood. To this end, we generated a set of melanoma and neuroblastoma cell lines stably overexpressing wild-type (WT), catalytically inactive (D508N) and N-terminal mutants, or shRNA against ATP13A2. We found that under proteotoxic stress conditions, evoked by the proteasome inhibitor Bortezomib, endo-/lysosomal associated full-length ATP13A2 WT, catalytically-inactive or N-terminal fragment mutants, reduced the intracellular accumulation of ubiquitin-conjugated (Ub) proteins, independent of autophagic degradation. In contrast, ATP13A2 silencing increased the intracellular accumulation of Ub-proteins, a pattern also observed in patient-derived fibroblasts harbouring ATP13A2 loss-of function mutations. In treated cells, ATP13A2 evoked endocytic vesicle relocation and increased cargo export through nanovesicles. Expression of an ATP13A2 mutant abrogating PI(3,5)P2 binding or chemical inhibition of the PI(3,5)P2-generating enzyme PIKfyve, compromised vesicular trafficking/nanovesicles export and rescued intracellular accumulation of Ub-proteins in response to proteasomal inhibition. Hence, our study unravels a novel activity-independent scaffolding role of ATP13A2 in trafficking/export of intracellular cargo in response to proteotoxic stress.
ATP13A2（也称为PARK9）是一种跨膜的与内体/溶酶体相关的P5型转运ATP酶。ATP13A2功能丧失性突变会导致库福-拉凯布综合征（KRS），这是常染色体显性帕金森病（PD）的一种形式。尽管人们对ATP13A2的兴趣日益浓厚，但对其在应激细胞中的生理作用却知之甚少。最近的研究表明，ATP13A2的N端结构域可能具有关键的调节功能，但其性质仍未完全了解。为此，我们构建了一组稳定过表达野生型（WT）、催化失活型（D508N）和N端突变体，或针对ATP13A2的短发夹RNA（shRNA）的黑色素瘤和神经母细胞瘤细胞系。我们发现，在蛋白酶体抑制剂硼替佐米引发的蛋白毒性应激条件下，与内体/溶酶体相关的全长ATP13A2 WT、催化失活型或N端片段突变体，减少了泛素共轭（Ub）蛋白的细胞内积累，且不依赖自噬降解。相反，ATP13A2沉默增加了Ub蛋白的细胞内积累，在携带ATP13A2功能丧失性突变的患者来源的成纤维细胞中也观察到了这种模式。在处理过的细胞中，ATP13A2引发内吞小泡重新定位，并通过纳米小泡增加货物输出。消除PI(3,5)P2结合的ATP13A2突变体的表达或对生成PI(3,5)P2的酶PIKfyve的化学抑制，损害了小泡运输/纳米小泡输出，并挽救了蛋白酶体抑制后Ub蛋白的细胞内积累。因此，我们的研究揭示了ATP13A2在响应蛋白毒性应激时，在细胞内货物运输/输出中一种新的不依赖活性的支架作用。
