Peroxidative aggregation of α‐synuclein requires tyrosines

Abstract

Abstract␣-Synuclein is the main component of the intracellular protein aggregates in neurons of patients with Parkinson's disease. The occurrence of the disease is associated with oxidative damage. Although it is known that peroxidative chemistry leads to the aggregation of ␣-synuclein in vitro, the specific amino acid types of ␣-synuclein involved in this type of aggregation have not been identified. We show, using human cytochrome c plus H 2 O 2 as the source oxidative stress, that the tyrosines of ␣-synuclein are required for aggregation. The studies reveal the chemical basis for a crucial step in the aggregation process.Keywords: aggregation; Lewy bodies; oxidative stress; Parkinson's disease; ␣-synuclein; tyrosine Parkinson's disease is one of the most common age-related neurodegenerative disorders, affecting almost 3% of the population more than 65 years of age. This devastating disease, clinically characterized by tremor, rigidity, and bradykinesia, affects more than one million persons in the United States alone (Lang and Lozano 1998). The symptoms are caused by the loss of dopaminergic neurons in the substantia nigra region of the brain. The few surviving neurons in this region contain Lewy bodies (Holdorff 2002), intracytoplasmic aggregates composed largely of the protein ␣-synuclein (Spillantini et al. 1997).␣-Synuclein became the focus of Parkinson's disease research after the discovery of two rare, familial forms of the disease caused by point mutations in the ␣-synuclein gene (Polymerpoulos et al. 1997). Although the exact functions of ␣-synuclein are unknown, two main biochemical activities have been proposed: regulation of dopamine neurotransmission and regulation of dopaminergic synaptic vesicles. ␣-Synuclein is natively disordered (Weinreb et al. 1996) and has a random-coil circular dichroism spectrum (Kim 1997), but its ␣-helical content increases from 3% to 63%-70% on binding lipid membranes (Davidson et al. 1998).The cause of ␣-synuclein aggregation to form Lewy bodies is unknown. However, the production of reactive oxygen species (ROS) has been suggested as a central event. Oxidative stress is an intracellular imbalance in the pro-oxidant/ antioxidant equilibrium, favoring pro-oxidants (Sies 1991). The main source of intracellular ROS is the mitochondria (Boveris et al. 1972;Boveris and Chance 1973), where ROS are produced during the reduction of oxygen to water. Under conditions of oxidative stress, both ROS and the respiratory chain protein cytochrome c can leak from the mitochondria into the cytoplasm (Shigenaga et al. 1994). It has been suggested that this leakage is facilitated by pores in the mitochondrial membranes formed by ␣-synuclein (Lashuel et al. 2002;Volles and Lansbury Jr. 2002). Whatever the mechanism, cytochrome c co-localizes with ␣-synuclein in Lewy bodies (Hashimoto et al. 1999).All cellular components are vulnerable to oxidative stress. The damage to proteins includes side chain modification and main chain fragmentation. Two biological markers for oxidative damage in pr...

Peroxidase Mechanism of Lipid-dependent Cross-linking of Synuclein with Cytochrome c

“…because of the lack of reactive and accessible Tyr residues in a protein). Notably, the presence of Tyr residues is essential for aggregation of Syn and LB formation as well as for its involvement in cross-linking through a catalytic peroxidase process (53), in line with our findings on protein-derived (Tyr ⅐ ) radicals in oligomers and high molecular weight aggregates.…”

Biophysical properties and cellular toxicity of covalent crosslinked oligomers of α-synuclein formed by photoinduced side-chain tyrosyl radicals

“…Several in vitro studies of aS have shown the relevance of tyrosine residues to the oxidation-induced formation of covalent aggregates, in which stable covalent interprotein -C-C-bonds in the form of dityrosine (diTyr) groups are implicated [11,12]. DiTyr has also been identified as an oxidative stress marker in brain tissue models of PD [13].…”

“…Considering that oxidative stress is one of the major factors linked to PD, oxidation of aS residues is a pathway to be considered. In previous studies, the specific oxidation of side-chain tyrosine of aS was achieved by peroxidative or peroxynitrile chemistry, leading to the formation of n-mers (n Z2) as well as lower molecular breakdown products [11,12,15,26,27]. Other oxidative stress models based on Fenton chemistry (i.e., a transition metal/H 2 O 2 or enzyme/H 2 O 2 systems) have also been extensively employed to reproduce in vitro aS covalent aggregation, but these systems are less specific in that they induce other modifications of amino acid residues, as in the case of Cu 2 þ þH 2 O 2 [28].…”

“…Other oxidative stress models based on Fenton chemistry (i.e., a transition metal/H 2 O 2 or enzyme/H 2 O 2 systems) have also been extensively employed to reproduce in vitro aS covalent aggregation, but these systems are less specific in that they induce other modifications of amino acid residues, as in the case of Cu 2 þ þH 2 O 2 [28]. In contrast, the involvement of tyrosine is essential in the peroxidative aggregation of aS induced by H 2 O 2 þ cytochrome c, but this process also yields aS degradation products and cytochrome c-aS adduct species [11]. In summary, the nature of the sources of reactive oxygen or nitrogen species (ROS and RNS, respectively) is a key feature of oxidation-induced aggregation.…”

Protein Folding, Unfolding and Misfolding: Role Played by Intermediate States

“…An additional role for the peroxidase activity of cyt c has been recently proposed in the study of the oxidative stressinduced aggregation of -synuclein, a "natively unfolded" protein constituting the major component of intracellular inclusions in several neurodegenerative disorders, such as Parkinson's disease [82,83]. Recently, some authors have observed the formation of millimeter-length fibers of phospholipid vesicles-bound cyt c displaying amyloid (ß-sheet) characteristics [84]; this implies that the cyt c-phospholipids interaction may play a role in some of the disorders attributed to amyloid formation, such as AA-amyloidosis and Alzheimer's disease.…”