Oxidative stress appears to be directly involved in the pathogenesis of several neurodegenerative disorders, including Alzheimer and Parkinson diseases. Nigral dopaminergic neurons are particularly exposed to oxidative stress because a pathological accumulation of cytosolic dopamine gives rise to various toxic molecules, including free radicals and reactive quinones. These latter species can react with proteins preventing them from exerting their physiological functions. Among the possible targets of quinones, ␣-synuclein is of primary interest because of its direct involvement in dopamine metabolism. Contrary to the neurotoxic processes, neuromelanin synthesis seems to play a protective role by its ability to sequester a variety of potentially damaging substances. In this study, we carried out a kinetic and structural analysis of the early oxidation products of dopamine. Specifically, considering the potential high toxicity of aminochrome for both cells and mitochondria, we focused our attention on its rearrangement to 5,6-dihydroxyindole. After the spectroscopic characterization of the products derived from the oxidation of dopamine, the structural information obtained was used to analyze the reactivity of quinones toward ␣-synuclein. Our results suggest that indole-5,6-quinone, rather than dopamine-o-quinone or aminochrome, is the reactive species. We propose that the observed reactivity could represent a general reaction pathway whenever cysteinyl residues are absent in proteins or if they are sterically protected.Parkinson disease, the second most common neurodegenerative disorder, is a chronic and progressive disease characterized by degeneration of dopaminergic neuromelanin-containing neurons in the substantia nigra pars compacta (1) and by the presence of cytoplasmic inclusions that are mainly composed of fibrillar ␣-synuclein (␣syn) 2 (2). Postmortem studies support the involvement of oxidative stress and the production of reactive oxygen species in Parkinson disease (3, 4). A possible source of oxidative stress is the redox reactions that specifically involve dopamine (DA). A critical aspect is the amount of DA present in the cytoplasm, outside the synaptic vesicles where the neurotransmitter is confined under physiological conditions. Spontaneous oxidation of DA in the presence of molecular oxygen leads to the formation of several cytotoxic molecules, including superoxide anions (O 2 . ), hydroxyl radicals (OH ⅐ ), and reactive quinones (DAQs) (5). Reactive oxygen species derived from the oxidation of DA can damage cellular components such as lipids, proteins, and DNA (6). The electrondeficient quinones can also react with cellular nucleophiles, leading to further cytotoxicity. DAQs have been shown to bind covalently to cysteinyl residues of proteins both in vitro and in vivo (7-12). Because these residues are often located at the active site of a protein, it has been proposed that covalent modifications result in an impairment of protein function with potentially deleterious effects on the cell (10...

Human alpha-synuclein is a 140-amino acid protein of unknown function abundantly expressed in the brain and found in Lewy bodies, a characteristic feature of Parkinson's disease. Alpha-synuclein is random in water under physiological conditions, but the first approximately 100 residues interact with SDS micelles or acidic phospholipid small unilamellar vesicles and adopt an ordered conformation. The rest of the molecule remains disordered in the bulk of the solution. The conformation of the N-terminal portion of the molecule in lipids was described as an extended helix [Ramakrishnan, M., Jensen, P. H., and Marsh, D. (2003) Biochemistry 42, 12919-12926], as two distinct alpha-helices interrupted by a two-residue break [Chandra, S., Chen, X., Rizo, J., Jahn, R., and Sudhof, T. C. (2003) J. Biol. Chem. 278, 15313-15318], or as a noncanonical conformation, the alpha11/3 helix [Bussell, R., Jr., and Eliezer, D. (2003) J. Mol. Biol. 329, 763-778]. We characterized the topology of the different regions of alpha-synuclein relative to the surface of SDS micelles using spin probe-induced broadening of NMR signals, (15)N relaxation measurements, and fluorescence spectroscopy. Our results support the presence of two N-terminal helices, positioned on the surface of the micelle and separated by a flexible stretch. The region of residues 61-95 of the protein also adopts a helical conformation, but it is partially embedded in the micelle. These results could shed some light on the role of the membrane on the aggregation process of alpha-synuclein.

Parkinson’s disease is a neurodegenerative disorder characterized by the death of dopaminergic neurons and by accumulation of alpha-synuclein (aS) aggregates in the surviving neurons. The dopamine catabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is a highly reactive and toxic molecule that leads to aS oligomerization by covalent modifications to lysine residues. Here we show that DOPAL-induced aS oligomer formation in neurons is associated with damage of synaptic vesicles, and with alterations in the synaptic vesicles pools. To investigate the molecular mechanism that leads to synaptic impairment, we first aimed to characterize the biochemical and biophysical properties of the aS-DOPAL oligomers; heterogeneous ensembles of macromolecules able to permeabilise cholesterol-containing lipid membranes. aS-DOPAL oligomers can induce dopamine leak in an in vitro model of synaptic vesicles and in cellular models. The dopamine released, after conversion to DOPAL in the cytoplasm, could trigger a noxious cycle that further fuels the formation of aS-DOPAL oligomers, inducing neurodegeneration.

Escherichia coli ribosomal protein S1 is required for the translation initiation of messenger RNAs, in particular when their Shine–Dalgarno sequence is degenerated. Closely related forms of the protein, composed of the same number of domains (six), are found in all Gram-negative bacteria. More distant proteins, generally formed of fewer domains, have been identified, by sequence similarities, in Gram-positive bacteria and are also termed ‘S1 proteins’. However in the absence of functional information, it is generally difficult to ascertain their relationship with Gram-negative S1. In this article, we report the solution structure of the fourth and sixth domains of the E. coli protein S1 and show that it is possible to characterize their β-barrel by a consensus sequence that allows a precise identification of all domains in Gram-negative and Gram-positive S1 proteins. In addition, we show that it is possible to discriminate between five domain types corresponding to the domains 1, 2, 3, 4–5 and 6 of E. coli S1 on the basis of their sequence. This enabled us to identify the nature of the domains present in Gram-positive proteins and, subsequently, to probe the filiations between all forms of S1.