Mutations in the GBA1 gene, encoding the enzyme glucocerebrosidase (GCase), cause the lysosomal storage disorder, Gaucher disease (GD), and are associated with the development of Parkinsons disease (PD) and other Lewy body disorders. Interestingly, GBA1 variants are the most common genetic risk factor associated with PD. While clinical studies argue a strong case towards a link between GBA1 mutations and the development of PD, mechanistic insights have been lacking. Recent research suggests a relationship between GCase and the PD-related amyloid-forming protein, alpha-synuclein;however, the specific molecular mechanisms responsible for association remain elusive. While the effects of GCase on alpha-synuclein homeostasis are the subject of considerable work, a role for alpha-synuclein in enzyme function has not been established. Such information could have further implications and indicate other mechanisms responsible for the increased PD risk. In prior work, we showed that alpha-synuclein and GCase interact selectively under lysosomal conditions, and proposed that this newly identified interaction might influence cellular levels of alpha-synuclein by either promoting protein degradation and/or inhibiting aggregation. We now extend our study to consider how membranes would modulate this complex formation. Lipids function not only as the substrate for GCase in intralysosomal vesicles, but also a surface for alpha-synuclein conformation alteration. For the first time, we demonstrate that the two proteins associate both on and off the membrane with comparable affinity, with apparent dissociation constants in the micromolar range. Using site-specific fluorescence and Forster energy transfer probes, we mapped the protein-enzyme interacting regions on unilamellar vesicles. Our data suggest that on the membrane surface, the GCase-alpha-synuclein interaction involves a larger alpha-synuclein region compared to that found in solution. Binding of alpha-synuclein to the membrane is critical for complex formation. In the absence of the N-terminal membrane binding domain, alpha-synuclein and GCase do not associate on the membrane, whereas in solution, this complex stays intact. Due to the likelihood of electrostatic repulsion between the negatively charged membrane surface and the acidic C-terminal tail, we suggest that at least some of the N-terminal membrane-binding residues, 1-95, are required to anchor alpha-synuclein to the membrane, thereby promoting and/or stabilizing its binding to GCase at the C-terminal. Despite the current interest in defining the role of GCase in PD, our study is the first to investigate whether alpha-synuclein has a direct effect on GCase function. We show that alpha-synuclein is a potent GCase inhibitor (an apparent IC50 in the submicromolar range) only when it adopts an alpha-helical conformation, supporting the concept that not only is membrane binding critical in modulating activity, but it also affects the specific conformational change of alpha-synuclein upon lipid and enzyme association. The observed mixed mode of inhibition suggests that at the membrane interface, alpha-synuclein binding influences both substrate accessibility and turnover. This study supports the notion that GCase deficiency can contribute to the pathogenesis of the synucleinopathies. The observed interplay between alpha-synuclein-GCase association and activity can be a potential self-perpetuating mechanism connecting enzyme deficiency and the accumulation of alpha-synuclein and substrate. This link between membrane-bound alpha-synuclein and GCase could be one of many possible mechanisms that connect GD and PD. However, since only a minority of GD patients or carriers develops PD, other pathological factors are likely involved. Nevertheless, it is now possible to search for proteins and other molecules that could modulate this alpha-synuclein-GCase interaction and to evaluate their effects on enzyme activity.

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Project End
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Budget End
Support Year
2
Fiscal Year
2013
Total Cost
$397,740
Indirect Cost
Name
National Heart, Lung, and Blood Institute
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Type
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Yap, Thai Leong; Velayati, Arash; Sidransky, Ellen et al. (2013) Membrane-bound ?-synuclein interacts with glucocerebrosidase and inhibits enzyme activity. Mol Genet Metab 108:56-64