Membrane-bound α-synuclein interacts with glucocerebrosidase and inhibits enzyme activity

Abstract

Mutations in GBA, the gene encoding glucocerebrosidase, the lysosomal enzyme deficient in Gaucher disease increase the risk for developing Parkinson disease. Recent research suggests a relationship between glucocerebrosidase and the Parkinson disease-related amyloid-forming protein, α-synuclein; however, the specific molecular mechanisms responsible for association remain elusive. Previously, we showed that α-synuclein and glucocerebrosidase interact selectively under lysosomal conditions, and proposed that this newly identified interaction might influence cellular levels of α-synuclein by either promoting protein degradation and/or preventing aggregation. Here, we demonstrate that membrane-bound α-synuclein interacts with glucocerebrosidase, and that this complex formation inhibits enzyme function. Using site-specific fluorescence and Förster energy transfer probes, we mapped the protein-enzyme interacting regions on unilamellar vesicles. Our data suggest that on the membrane surface, the glucocerebrosidase-α-synuclein interaction involves a larger α-synuclein region compared to that found in solution. In addition, α-synuclein acts as a mixed inhibitor with an apparent IC(50) in the submicromolar range. Importantly, the membrane-bound, α-helical form of α-synuclein is necessary for inhibition. This glucocerebrosidase interaction and inhibition likely contribute to the mechanism underlying GBA-associated parkinsonism.

Fig 1

α-Syn GCase interaction in the presence of vesicles. A, GCase crystal structure (PDB code 2NSX [60]). The intrinsic 12 Trp residues are shown in gray and were used as Förster energy transfer donors. Active site residues (E235 and E340) are in yellow. Loops 1–3, regions proposed to be important for substrate recognition and activity [60, 61], are highlighted in blue. For clarity, residues 286–290 were omitted. B, Schematic representation of α-syn primary sequence. The seven amphipathic imperfect repeat regions (blue) in the membrane binding domain (gray) and the acidic C-terminal region (red) are denoted. The location of Dns-labeling sites, residues 7, 40 and 57 (membrane binding domain) and residues 100 and 136 (C-terminal domain) are indicated. The same color representation is used in panels C and D. Location of peptide C23 (residue 118–140) is also indicated. C, Normalized change in mean residue ellipticity Δ[θ]) as a function of lipid-to-protein ratio (50 mM MES, 25 mM NaCl, pH 5.5, [WT α-syn] = 5 μM). Error bars indicate standard deviations from two independent measurements (top left). POPC/POPS vesicles induced secondary structure in α-syn (top left, inset). GCase titration curves for Dns136-α-syn (top right), Dns100-α-syn (bottom left), and Dns136-C23 peptide (bottom right). The titration curves were obtained from mean spectral wavelength (<λ>) in 50 mM MES, 25 mM NaCl at pH 5.5 in the absence (●) or presence of 0.9 mM POPC/POPS vesicles (■). Error bars indicate standard deviations from two replicate samples. Fits are shown as lines. D, Förster energy transfer from GCase to DnsX-α-syn. Measurements were performed in the presence of 2 μM GCase (Trp donor, excited at 295 nm) and 5 μM DnsX-α-syn (Dns acceptor), either in the absence or presence of vesicles at pH 5.5 (colored according to panel B). Fluorescence spectra of GCase and DnsX-α-syn alone are shown in gray and green, respectively. WT syn was used as a negative control.

Fig 2

α-Syn inhibits GCase activity. A, Relative GCase activity as a function of added α-, β-, and γ-synuclein in the presence of 350 μM POPC/POPS vesicles ([GCase] = 50 nM and [4MU-β-glu] = 1 mM, IC₅₀ ~215, 850, and 685 nM, respectively). Top Inset, Effect of α-syn on GluCer turnover by GCase ([GCase] = 50 nM and 350 μM POPC/POPS/GluCer vesicles). Bottom, Relative GCase activity as a function of added α-syn in the presence of 350 μM POPC/BMP vesicles ([GCase] = 50 nM and [4MU-β-glu] = 1 mM, IC₅₀ ~230 nM). B, Comparison of amino acid sequences of α-, β-, and γ-synuclein. N-terminal residues (1–44) were omitted as the sequences are nearly identical (95.5 and 84% for β and γ-syn, respectively). Differences are colored in red and the residue is colored yellow if the side chain change retains similar charge. Alignment was made using the program ALIGN available at www.uniprot.org/align/

Fig 3

Effect of α-syn secondary structure on GCase activity. A, Comparison of GCase activity in the presence of NaTc (black) and POPC/POPS vesicles (gray) upon the addition of α-syn. B, GCase titration curve obtained from mean spectral wavelength (<λ>) of Dns136-α-Syn at pH 5.5 in the presence of 3 mM NaTc. Fit is shown as a line. Inset, Circular dichroism spectra of α-syn in the presence of NaTc (black) and POPC/POPS vesicles (gray). C, A schematic diagram depicting that while α-syn interacts with GCase in the presence of both NaTc and POPC/POPS vesicles, only the α-helical vesicle-bound α-syn inhibits enzyme activity.

Fig 4

Mechanism of inhibition by α-syn. A, GCase (10 nM) reaction velocity as a function of added 4MU-β-glu in the presence of 0 μM (●), 0.15 μM (■), 0.3 μM (▲) and 1.25 μM (▼) α-syn. Fits to Michaelis-Menten equation for mixed inhibition are shown as lines. B, Plot of apparent V_m(app) vs. K_m(app) extracted from fits.

Fig 5

Schematic model of the α-syn–GCase complex at the membrane interface. Left, View from the top. Membrane binding and α-helical domains of α-syn (residues 1–37 and 51–92) are represented as cylinders and connected by a flexible linker. The disordered C-terminal tail is drawn black. This open horseshoe structure is adapted from the sodium dodecyl sulfate micelle-bound structure (PDB code 1XQ8 [53]). The five fluorescent probes are colored coded to indicate membrane-binding (blue: Dns7 and Dns40), GCase-binding (red: Dns100 and Dns136) and membrane-and-GCase binding (purple: Dns57). In this model, α-syn residues 118–140 reside in the groove between domains I and III of GCase (See Fig. S9 for more detailed renderings). Right, View from the side. The GCase active site is represented by yellow triangle. The relative orientations (tilt) of the helices on the bilayer need not be identical since there is a flexible hinge connecting them.

Fig 6

Molecular links between glucocerebrosidase and α-synuclein. In normal functional lysosomes, wild-type GCase interacts with α-syn, facilitating degradation. In some cases, mutant and/or decreased levels of wild-type GCase increase the probability of α-syn accumulation, as well as the buildup of glucocerebroside (GluCer). The inhibitory effect of α-syn may lead to a secondary loss of enzyme activity, which would further compound the problem.