The Impact of the E46K Mutation on the Properties of α-Synuclein in Its Monomeric and Oligomeric States

Abstract

The third and most recently identified Parkinson's disease-linked variant of the neuronal protein R-synuclein to be identified (E46K) results in widespread brain pathology and early onset Parkinson symptoms (Zarranz et al. (2004) Ann. Neurol. 55, 164-173). Herein, we present biochemical and biophysical characterization of E46K R-synuclein in various states of aggregation. Circular dichroism and nuclear magnetic resonance spectroscopy illustrate that the E46K mutation results in subtle changes in the conformation of the monomeric protein both free in solution and in the presence of SDS micelles. However, it does not alter the overall helical propensity of the protein in the presence of phospholipids. E46K R-synuclein formed insoluble fibrils in Vitro more rapidly than the wild type protein, and electron microscopy revealed that E46K R-synuclein fibrils possess a typical amyloid ultrastructure. E46K R-synuclein protofibrils, soluble aggregates that form during the transition from the monomeric form to the fibrillar form of R-synuclein, were characterized by electron microscopy and gel filtration and were found to include annular species. The unique ability of a subfraction of E46K and wild type R-synuclein protofibrils containing porelike species to permeabilize lipid vesicles was demonstrated in Vitro using a real-time chromatographic method. In contrast to simplistic expectations, the total amount of protofibrils and the amount of permeabilizing activity per mole protein in the protofibril fraction were reduced by the E46K mutation. These results suggest that if the porelike activity of R-synuclein is important for neurotoxicity, there must be factors in the neuronal cytoplasm that reverse the trends in the intrinsic properties of E46K versus WT R-synuclein that are observed in Vitro.Parkinson's disease (PD 1 ) is a progressive neurodegenerative disorder characterized by resting tremor, bradykinesia, rigidity, and postural instability due to the selective loss of dopaminergic neurons within the substantia nigra (2, 3). While nearly all cases of PD are idiopathic, rare forms of autosomal dominant PD have been linked to the point mutations A53T (4), A30P (5), and E46K (1) in R-synuclein, a presynaptic protein believed to be involved in synaptic vesicle trafficking (6,7). Linkage between idiopathic PD and R-synuclein is suggested by the discovery that Lewy bodies (LB), intraneuronal cytoplasmic inclusions in the substantia nigra that are the pathological hallmark of PD, are composed primarily of fibrillar R-synuclein (8).Monomeric R-synuclein is natively unfolded in solution (9), but assumes -sheet character as it aggregates through a series of intermediate, metastable oligomeric states (termed protofibrils) to a stable fibrillar conformation (10). In Vitro studies have shown that both the A53T and A30P mutations in R-synuclein alter the kinetics of fibrillization; the rate is increased for the A53T variant and retarded by the A30P substitution (11,12). However, in both cases the rates of formation of pre...

Identification of a helical intermediate in trifluoroethanol-induced alpha-synuclein aggregation

“…Thus, secondary structural transitions appear to be largely similar among αS mutants. In contrast, oligomerization and fibrillization behavior vary significantly between PD-linked mutants, with amyloid fibril formation rates observed in the order A30P < WT < A53T∕ E46K (23,35). Likewise, we find that TFE-induced oligomerization rates vary significantly among the αS variants despite their nearly identical monomer secondary structure landscapes.…”

“…We find that the TFE-induced folding landscapes for the A30P, A53T, and E46K mutants are nearly identical to WT αS, which is in accordance with previous research that showed that the pH-and temperature-induced secondary structural conformations are similar for A30P, A53T, and WT αS (34). All three mutants have also been observed to undergo similar structural transitions to WT αS in the presence of detergents or lipids (35)(36)(37), although the A30P mutation may lead to a slight local reduction in helical structure. Thus, secondary structural transitions appear to be largely similar among αS mutants.…”

Oxidative Stress and Neurotoxicity

“…Point mutations in R-synuclein that characterize the rare heritable forms of PD have been seen to increase the rate of formation of either fibrils or protofibril intermediates (205 (208), or nitrating reagents (209) induce aggregation/fibrillization of the protein, and human Lewy bodies and other R-synuclein inclusions are positive to antinitrotyrosine antibodies (210). However, oxidation of the four Met residues in R-synuclein to MetO can completely inhibit fibrillization of the peptide if there are no metals around (211).…”

The phosphorylation state of Ser-129 in human α-synuclein determines neurodegeneration in a rat model of Parkinson disease

“…Studies have suggested that ␣-syn can form soluble oligomers and fibrillar species that may be precursors to Lewy bodies and are associated with the neurotoxicity seen in animals and humans. A number of studies have suggested that soluble oligomers or protofibrils are likely to be the toxic species and that fibrils and Lewy bodies may be protective by sequestering excess ␣-syn (4,5).…”