Protection against Mitochondrial and Metal Toxicity Depends on Functional Lipid Binding Sites in ATP13A2.

Shaun Martin,Veerle Baekelandt,Jan Eggermont,Tine Holemans,Peter Vangheluwe,Sarah van Veen,Chris van den Haute,Seyma Demirsoy,Patrizia Agostinis

doi:10.1155/2016/9531917

Abstract

The late endo-/lysosomal P-type ATPase ATP13A2 (PARK9) is implicated in Parkinson's disease (PD) and Kufor-Rakeb syndrome, early-onset atypical Parkinsonism. ATP13A2 interacts at the N-terminus with the signaling lipids phosphatidic acid (PA) and phosphatidylinositol (3,5) bisphosphate (PI(3,5)P2), which modulate ATP13A2 activity under cellular stress conditions. Here, we analyzed stable human SHSY5Y cell lines overexpressing wild-type (WT) or ATP13A2 mutants in which three N-terminal lipid binding sites (LBS1–3) were mutated. We explored the regulatory role of LBS1–3 in the cellular protection by ATP13A2 against mitochondrial stress induced by rotenone and found that the LBS2-3 mutants displayed an abrogated protective effect. Moreover, in contrast to WT, the LBS2 and LBS3 mutants responded poorly to pharmacological inhibition of, respectively, PI(3,5)P2 and PA formation. We further demonstrate that PA and PI(3,5)P2 are also required for the ATP13A2-mediated protection against the toxic metals Mn2+, Zn2+, and Fe3+, suggesting a general lipid-dependent activation mechanism of ATP13A2 in various PD-related stress conditions. Our results indicate that the ATP13A2-mediated protection requires binding of PI(3,5)P2 to LBS2 and PA to LBS3. Thus, targeting the N-terminal lipid binding sites of ATP13A2 might offer a therapeutic approach to reduce cellular toxicity of various PD insults including mitochondrial stress.

Highlights

Mitochondria are organelles with a pivotal role in ATP production, intracellular Ca2+ signaling, the generation of reactive oxygen species (ROS), and apoptotic cell death [1,2,3]
Using purified N-terminal fragments of ATP13A2 in protein lipid overlay assays, we described that LBS1 and LBS2 are mainly required for interaction with propidium iodide (PI)(3,5)P2 (PA to a lesser extent), whereas LBS3 is essential for phosphatidic acid (PA) interaction [14] (Figure 1(a))
Our results show that both PI(3,5)P2 and PA are required to exert ATP13A2-mediated cellular protection, which is explained by a specific and direct interaction of PI(3,5)P2 and PA at the ATP13A2 N-terminal lipid binding sites LBS2 and LBS3, respectively

Summary

Introduction

Mitochondria are organelles with a pivotal role in ATP production, intracellular Ca2+ signaling, the generation of reactive oxygen species (ROS), and apoptotic cell death [1,2,3]. Because of high energy demands at locations distant from the cell body, neurons in particular critically depend on healthy and dynamic mitochondria to fuel membrane excitability and to execute neurotransmission and plasticity [4, 5]. PD is hallmarked by the accumulation of aggregated α-synuclein into Lewy bodies in neurons of the substantia nigra and specific brain stem, spinal cord, and cortical regions [7, 8], and mitochondrial defects are common [9,10,11]. Several PD-associated genes, mainly parkin and PINK1, play a role in mitochondrial dynamics and clearance via mitophagy, which strengthens

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