Membrane insertion exacerbates the α-Synuclein-Cu(II) dopamine oxidase activity: Metallothionein-3 targets and silences all α-synuclein-Cu(II) complexes

Abstract

Copper binding to α-synuclein (α-Syn), the major component of intracellular Lewy body inclusions in substantia nigra dopaminergic neurons, potentiate its toxic redox-reactivity and plays a detrimental role in the etiology of Parkinson disease (PD). Soluble α-synuclein-Cu(II) complexes possess dopamine oxidase activity and catalyze ROS production in the presence of biological reducing agents via Cu(II)/Cu(I) redox cycling. These metal-centered redox reactivities harmfully promote the oxidation and oligomerization of α-Syn. While this chemistry has been investigated on recombinantly expressed soluble α-Syn, in vivo, α-Syn is acetylated at its N-terminus and is present in equilibrium between soluble and membrane-bound forms. This post-translational modification and membrane-binding alter the Cu(II) coordination environment and binding modes and are expected to affect the α-Syn-Cu(II) reactivity. In this work, we first investigated the reactivity of acetylated and membrane-bound complexes, and subsequently addressed whether the brain metalloprotein Zn₇-metallothionein-3 (Zn₇MT-3) possesses a multifaceted-role in targeting these aberrant copper interactions and consequent reactivity. Through biochemical characterization of the reactivity of the non-acetylated/N-terminally acetylated soluble or membrane-bound α-Syn-Cu(II) complexes towards dopamine, oxygen, and ascorbate, we reveal that membrane insertion dramatically exacerbates the catechol oxidase-like reactivity of α-Syn-Cu(II) as a result of a change in the Cu(II) coordination environment, thereby potentiating its toxicity. Moreover, we show that Zn₇MT-3 can efficiently target all α-Syn-Cu(II) complexes through Cu(II) removal, preventing their deleterious redox activities. We demonstrate that the Cu(II) reduction by the thiolate ligands of Zn₇MT-3 and the formation of Cu(I)₄Zn₄MT-3 featuring an unusual redox-inert Cu(I)₄-thiolate cluster is the molecular mechanism responsible for the protective effect exerted by MT-3 towards α-Syn-Cu(II). This work provides the molecular basis for new therapeutic interventions to control the deleterious bioinorganic chemistry of α-Syn-Cu(II).

Keywords: Alpha-synuclein; Copper dysregulation; Dopamine oxidation; Metallothionein-3; Parkinson's disease; Reactive oxygen species.

Figure 1.

Proposed copper coordination modes in α-Syn. (a) Cu(II) coordination at the high-affinity N-terminal site in soluble α-Syn at physiological pH; (b) Cu(I) coordination at the N-terminal site; (c) high affinity N-terminal site in membrane-bound form; (d) ^NAcα-Syn high-affinity site centered at His50; and (E) low-affinity C-terminal site.

Figure 2.

(a) Absorption spectra recorded after the reaction of ^NH2α-Syn-Cu(II) (10 μM; black), ^NAcα-Syn-Cu(II) (10 μM; red), or free Cu(II) (10 μM; gray) with dopamine (1 mM) in 20 mM N-ethylmorpholine/100 mM NaCl, pH 7.4 at 25°C, in the presence of MBTH (2 mM). Auto-oxidation of dopamine (1 mM) is plotted in pink. (b) Concentration-dependent dopamine oxidase activity of ^NH2α-Syn-Cu(II) (10 μM; black) or ^NAcα-Syn-Cu(II) (10 μM; red) in 20 mM N-ethylmorpholine/100 mM NaCl, pH 7.4, determined using MBTH (2 mM) to quantify the dopamine ortho-quinone formed after 120 s reaction (25°C).

Figure 3.

(a) Ascorbate-driven hydroxyl radical production by Cu(II) (5 μM; gray), ^NH2α-Syn-Cu(II) (5 μM; black), or ^NAcα-Syn-Cu(II) (5 μM; red) in the presence of ascorbate (600 μM) and 3-CCA (400 μM), determined by monitoring the formation of the fluorescent product 7-OH-CCA for 450 s at 37°C (λ_ex=395 nm; λ_em=450 nm). (b) Dityrosine emission spectra (λ_ex=325 nm) recorded for^NH2α-Syn-Cu(II) (10 μM; green and black) and ^NAcα-Syn (10 μM; orange and red) before and after 1-h reaction with 1 mM ascorbate (37°C). (c) Kinetic traces monitoring dityrosine formation at 418 nm (λ_ex=325 nm) in ^NH2α-Syn (green), ^NH2α-Syn-Cu(II) (black), ^NAcα-Syn (orange), and ^NAcα-Syn-Cu(II) (red). Dityrosine formation is reported as the difference between final and initial fluorescence at 418 nm (ΔF) over the initial fluorescence (F_i).

Figure 4.

(a) Size exclusion chromatogram monitoring membrane insertion of soluble ^NH2α-Syn (15 μM; black, blue after insertion) or ^NAcα-Syn (red, green after insertion) using a Superdex 200 column. Lipids (gray) elute at the void volume (~8.2 ml) while soluble α-Syn elutes at 13.3 ml (black and red). Inset: Enlarged portion of the chromatogram showing soluble α-Syn elution. (b) SDS-PAGE of membrane-bound ^NH2α-Syn and ^NAcα-Syn, generated after removing soluble protein using 50-kDa MWCO filters (SN: supernatant; FT: filtrate). (c) Dynamic light scattering size distribution analysis of lipid vesicles (0.1 mg/ml lipids) generated by sonication, indicating the formation of SUVs.

Figure 5.

(a) Concentration-dependent dopamine oxidase activity of soluble or membrane-bound ^NH2α-Syn-Cu(II) (10 μM; black and blue, respectively) and ^NAcα-Syn-Cu(II) (10 μM; red and green, respectively) in 20 mM N-ethylmorpholine/100 mM NaCl, pH 7.4, determined using MBTH (2mM) to quantify the dopaquinone formed. Values were obtained after 120 s reaction for soluble forms and 20 s for the membrane-bound forms (25°C). (b) Concentration-dependent dopamine oxidase activity of membrane-bound ^NAcα-Syn-Cu(II) (10 μM) in 20 mM N-ethylmorpholine/100 mM NaCl, pH 7.4 at increasing H₂O₂ concentrations (0 – 1 M), determined using 2 mM MBTH to quantify the dopaquinone formation (20 s, 25°C). (c) Michaelis-Menten analysis to determine V_{max, H2O2} as a function of H₂O₂ concentrations.

Figure 6.

(a) Ascorbate-driven hydroxyl radical production of membrane-bound ^NH2α-Syn-Cu(II) (5 μM; blue), membrane-bound ^NAcα-Syn-Cu(II) (5 μM; green), or Cu(II) bound to lipids (5 μM; gray), determined in the presence of 600 μM ascorbate and 400 μM 3-CCA by monitoring the formation of fluorescent product 7-OH-CCA (λ_ex=395 nm; λ_em=450 nm; 450 s; 37°C.) (b) Ascorbate-driven dityrosine formation of membrane-bound ^NH2α-Syn-Cu(II) or ^NAcα-Syn-Cu(II) (5 μM), determined by monitoring dityrosine fluorescence at 418 nm (λ_ex=325 nm) upon incubation with 3 mM ascorbate for 3 h (37°C). Dityrosine formation is reported as the difference between final and initial fluorescence at 418 nm (ΔF) divided by initial fluorescence (F_i).

Figure 7.

(a) Catalytic dopamine (1 mM) oxidation by ^NH2α-Syn-Cu(II) or ^NAcα-Syn-Cu(II) (10 μM) in 20 mM N-ethylmorpholine/100 mM NaCl, pH 7.4, in the presence 2 mM MBTH (100 min, 25°C). The dopamine oxidase activity is quenched upon addition of Zn₇MT-3 (0.25 eq., 1 h) prior to the addition of dopamine. (b) Quenching of the dopamine oxidase activity of membrane-bound ^NH2α-Syn-Cu(II) or ^NAcα-Syn-Cu(II) (10 μM) upon reaction with Zn₇MT-3 (0.25 eq.). The dopaquinone formed was quantified after 20 s (25°C) using 2 mM MBTH.

Figure 8.

(a) Low-temperature (77 K) luminescence emission spectra of the products of the reaction (1 h; 25°C) between soluble and membrane-bound ^NH2α-Syn-Cu(II) (10 μM; black and blue, respectively) or ^NAcα-Syn-Cu(II) (10 μM; red and green, respectively) with Zn₇MT-3 (2.5 μM). The reference spectra of Cu(I)₄Zn(II)₄MT-3 (2.5 μM) is plotted for comparison (gray). (b) Emission lifetime determination of the product of reaction between membrane-bound ^NAcα-Syn-Cu(II) and Zn₇MT-3 at 425 nm (black) and 575 nm (red). Lifetime values were determined by fitting using single exponential decay function (gray and orange, respectively).

Figure 9.

(a) Quenching of ascorbate-driven hydroxyl radical production of 5 μM soluble ^NH2α-Syn-Cu(II) (black) and ^NAcα-Syn-Cu(II) (red) by 0.25 eq. Zn₇MT-3 (green and orange lines, respectively) incubated for 1 h before reaction with 1 mM ascorbate. Hydroxyl radical production was monitored by following the formation of the fluorescent product 7-OH-CCA (λ_ex=395 nm; λ_em=450 nm) for 450 s (37°C). (b) Hydroxyl radical quenching by Zn₇MT-3 (1.25 μM) in the membrane-bound α-Syn-Cu(II) forms (5 μM). (c) Quenching of ascorbate-driven dityrosine formation of soluble ^NH2α-Syn-Cu(II) (black) and ^NAcα-Syn-Cu(II) (red) by Zn₇MT-3 (green and orange lines, respectively), incubated for 1 h before reaction was initiated by addition of ascorbate (1 mM). Dityrosine formation was determined by monitoring dityrosine fluorescence at 418 nm (λ_ex=325 nm) after 1 h reaction (37°C), reported as the difference between final and initial fluorescence (ΔF) divided by initial fluorescence (F_i). (d) Quenching of dityrosine formation by Zn₇MT-3 (1.25 μM) in the membrane-bound α-Syn-Cu(II) forms (5 μM), after following the reaction for 3 h upon the addition of ascorbate (3 mM). Values are corrected for ΔF/F_{i, 418nm} of membrane-bound α-Syn forms in the absence Cu(II).