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Title: Unlocking the transport activity of the P5-type transport ATPases in neurodegeneration
Other Titles: Het ontrafelen van de transport activiteit van de P5-type transport ATPasen in neurodegeneratie
Authors: Holemans, Tine
Issue Date: 18-Oct-2016
Abstract: The P5‐type ATPases are a subfamily belonging to the large family of P‐type transporters and are probably the least characterized. One of the most intriguing members is ATP13A2, a lysosomal membrane protein in which mutations underlie various neurological disorders, including Kufor‐Rakeb syndrome, Neuronal Ceroid Lipofuscinosis and Early‐Onset Parkinson’s Disease. It has previously been described that ATP13A2 deficiency is involved in a plethora of lysosomal and mitochondrial dysfunctions and numerous substrates have been proposed to be transported by the pump, varying from ions such as Mn2+ or Zn2+ to lipids, as is seen in the P4 ATPase subfamily.
Based on sequence comparison the P5 ATPases can be divided into two distinct groups, the P5A (ATP13A1) and the P5B (ATP13A2‐5) and this subdivision further reflects in various other characteristics. Indeed, we demonstrated the P5A ATPases to be localized in the endoplasmic reticum, whereas the P5B associate with the endo‐/lysosomal system. More specifically, ATP13A2 and ATP13A4 are targeted to the late endo‐/lysosomes, ATP13A3 is found in the early and recycling endosomes. We furthermore confirmed the predicted topology for the P5A, consisting of 12 transmembrane (TM) helices with the two N‐terminal helices Mb and Ma assembling a hairpin spanning the membrane. On the contrary, although the P5B were predicted to hold 11 TM helices, with N‐ and C‐terminal ends on opposite sides of the membrane, we refuted this topology and found the P5B to be built out of 10 TM helices and one additional N‐terminal helix, firmly associated with the membrane. For the first time, we established a biochemical assay for the P5B, defining the amount of phosphorylation. We could demonstrate the formation of a phospho‐intermediate for ATP13A1‐4, pointing to active enzymes. The assay now provides us with an indispensable tool for further research of the P5 ATPases and their disease mutants. It should be noted that it was very difficult to obtain consistent results for ATP13A5.
The presence of the additional membrane‐associated N‐terminal helix in ATP13A2, is a peculiar feature, which is entitled to be investigated more profound. Via protein‐lipid overlays, flotation assays and sequence mutagenesis, the N‐terminal helix Ma was shown to interact with two signaling lipids, i.e. phosphatidic acid (PA) and phosphatidylinositol(3,5)‐bisphosphate (PI(3,5)P2). Moreover, binding of these lipids activates the protein, which is otherwise pending in the E1P state, awaiting further activation. Markedly, the catalytic active enzyme encompasses an even more noticeable function as it provides protection against rotenone‐induced mitochondrial stress upon binding of PA and PI(3,5)P2. Depletion of one of these two lipids, abolishes the protective effect. This emphasizes the importance of the N‐terminus in this process and implicates the interaction of PA and PI(3,5)P2 with N‐terminus to offer a therapeutic strategy for protection against mitochondrial stress, one of the important distinctive features of Parkinson’s Disease or related disorders.
In spite of the fact that ATP13A2 is implicated in various disorders, no substantial biochemical evidence was present up until now. We here provided the first biochemical proof that disease mutations can indeed affect the catalytic activity of the pump as they display an impaired autophosphorylation. Finally, we also demonstrated ATP13A2 mutations to underlie a different neurological pathology, i.e. Hereditary Spastic Paraplegia.
For ATP13A2, it is becoming more and more clear that it plays an emerging and significant role in neurological disorders.
Table of Contents: Expression of gratitude 2
A. Content table 4
B. List of abbreviations 7
I. Introduction 9
1 P-type ATPases 9
1.1 Discovery and P-type ATPase phylogeny 10
1.1.1 Discovery of the P-type ATPase family 10
1.1.2 P-type ATPase phylogeny 11
1.1.2.1 P1-type ATPases 12
1.1.2.2 P2-type ATPases 12
1.1.2.3 P3-type ATPases 12
1.1.2.4 P4-type ATPases 13
1.1.2.5 P5-type ATPases 13
1.2 General characteristics of a P-type ATPase 13
1.2.1 The P-type ATPase architecture 13
1.2.2 The transport mechanism of P-type transport ATPases 15
2 P5-type ATPases: a branch still in its infancy 20
2.1 Subdivision into a P5A and a P5B group 20
2.1.1 The P5A P-type ATPases 20
2.1.1.1 Yeast SPF1 21
2.1.1.2 ATP13A1 in mammals 22
2.1.2 The P5B-type ATPases 23
2.1.2.1 Yeast YPK9 23
2.1.2.2 ATP13A2 24
2.1.2.3 ATP13A3-5 25
3 ATP13A2: Cellular role and pathophysiological implications 26
3.1 Pathological implications of ATP13A2 26
3.1.1 ATP13A2 in Parkinson’s Disease 26
3.1.2 ATP13A2 in Neuronal Ceroid Lipofuscinosis 27
3.2 Cellular function of ATP13A2 and connection to PD 27
3.2.1 ATP13A2 controls lysosomal functionality and mitochondrial health 27
3.2.2 ATP13A2, α-synuclein and other PD genes 29
3.3 The enigmatic substrate of ATP13A2 31
3.3.1 Is ATP13A2 a transporter of cations? 31
3.3.1.1 Is ATP13A2 transporting Mn2+? 32
3.3.1.2 Is ATP13A2 a Zn2+ transporter? 33
3.3.1.3 Is ATP13A2 transporting Mg2+? 34
3.3.2 Is ATP13A2 a lipid flippase involved in vesicular transport? 34
II. Objectives 36
III. Results 37
1 Molecular and biochemical properties of ATP13A2 37
2 Relationship between the P5-type ATPases 49
3 ATP13A2 in Hereditary Spastic Paraplegia 91
IV. Discussion 118
1 P5A and P5B ATPases, two groups with different properties and cellular functions 118
1.1 Phylogenetic and sequence analysis of P5A and P5B 118
1.2 Topology of P5A and P5B 119
1.3 Subcellular distribution of P5A and P5B 120
1.4 Autophosphorylation of P5A and P5B 122
2 Implications of the interaction of the ATP13A2 N-terminus with PA and PI(3,5)P2 123
3 ATP13A2 is involved in a spectrum of neurological disorders 126
4 The enigmatic substrate quest 127
4.1 P5-type ATPases as putative ion transporters 127
4.2 P5-type ATPases as putative lipid or organic molecule transporters 130
5 Future perspectives 131
5.1 Defining the transported substrate of the P5 ATPases 131
5.2 Test the complementation of the invertebrate P5B with the mammalian ATP13A1-5 isoforms in a small animal model 131
5.3 What is the specific molecular function of the N-terminus of the P5Bs? 132
5.3.1 What is the mechanism of the N-terminal autoinhibitory function? 132
5.3.2 Does the N-terminus exert a similar function as in the P1B ATPases? 132
5.3.3 Is a similar role reserved for the C-terminus of ATP13A2? 133
5.4 Can we establish the loss-of-function effect of various reported and novel ATP13A2 disease mutations? 133
V. Abstract 134
VI. References 136
VII. Curruculum Vitae 146
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Physiology Section (-)
Laboratory of Cellular Transport Systems

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