The Reaction of α-Synuclein with Tyrosinase: POSSIBLE IMPLICATIONS FOR PARKINSON DISEASE

Isabella Tessari,Marco Bisaglia,Francesco Valle,Bruno Samorì,Elisabetta Bergantino,Stefano Mammi,Luigi Bubacco

doi:10.1074/jbc.m709014200

Isabella Tessari, Marco Bisaglia + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m709014200

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Abstract

Oxidative stress appears to be directly involved in the pathogenesis of Parkinson disease. Several different pathways have been identified for the production of oxidative stress conditions in nigral dopaminergic neurons, including a pathological accumulation of cytosolic dopamine with the subsequent production of toxic reactive oxygen species or the formation of highly reactive quinone species. On these premises, tyrosinase, a key copper enzyme known for its role in the synthesis of melanin in skin and hair, has been proposed to take part in the oxidative chemistry related to Parkinson disease. A study is herein presented of the in vitro reactivity of tyrosinase with alpha-synuclein, aimed at defining the molecular basis of their synergistic toxic effect. The results presented here indicate that, in conformity with the stringent specificity of tyrosinase, the exposed tyrosine side-chains are the reactive centers of alpha-synuclein. The reactivity of alpha-synuclein depends on whether it is free or membrane bound, and the chemical modifications on the tyrosinase-treated alpha-synuclein strongly influence its aggregation properties. On the basis of our results, we propose a cytotoxic model which includes a possible new toxic role for alpha-synuclein exacerbated by its direct chemical modification by tyrosinase.

Highlights

Parkinson disease (PD)2 is the most common movement disorder and, after Alzheimer disease, the second most common neurodegenerative disorder affecting ϳ1–2% of people over 65 years
Tyrosinase, a key copper enzyme known for its role in the synthesis of melanin in skin and hair, has been proposed to take part in the oxidative chemistry related to Parkinson disease
PD is characterized by a striking loss of dopamine-producing neurons in the substantia nigra pars compacta, accompanied by depletion of dopamine in the striatum, and by the presence of cytoplasmic inclusions known as Lewy bodies

Summary

EXPERIMENTAL PROCEDURES

Protein Preparation and Purification—Human ␣Syn cDNA was amplified by PCR with synthetic oligonucleotides (SigmaGenosys) containing NcoI and XhoI restriction sites and designed to obtain the entire sequence of the protein (␣Syn140) or the region coding for the first 99 amino acids (␣Syn). Reaction Mixtures—The samples used in all the spectroscopic analyses contained ␣Syn140 or ␣Syn to a final concentration of 100 ␮M in the presence of 100 mM phosphate buffer (pH 7.4) and with the addition of the indicated quantity of S. antibioticus tyrosinase. Limited Proteolysis of ␣Syn by Chymotrypsin—A limited proteolysis experiment of ␣Syn in the presence of high purity chymotrypsin (Calbiochem) was carried out at 25 °C for 10 min in a reaction mixture containing 50 ␮g of protein and 1.5 units of chymotrypsin in 5–50 mM phosphate buffer (pH 7.4). Sample measurements were carried out using a HELLMA ultra-micro cell with Suprasil௡ windows and an optical path length of 10 ϫ 2 mm. All spectra were recorded in the 190 –260 nm wavelength range, using a bandwidth of 2 nm and a time constant of 2 s at a scan speed of 50 nm/min. Height relative to the surface, because this parameter is not influenced by the tip convolution effect

RESULTS

Molecular mass

Findings

DISCUSSION