Cleavage Agents for α-Synuclein

Abstract

Previously, we reported artificial proteases for soluble oligomers of amyloidogenic peptides such as amyloid β-42 (Aβ42) or human islet amyloid polypeptide (h-IAPP). Several reports suggest that soluble oligomers of Aβ42 and hIAPP are the pathogenic intermediates in Alzheimer’s disease and type 2 diabetes mellitus, respectively. Soluble oligomers of other amyloidogenic peptides or proteins (AmPs) such α-synuclein, prion, and polyglutamine are also implicated as the primary toxic species in amyloidoses such as Parkinson’s disease, spongiform encepahlopathies, and Huntington’s disease, respectively. The association process of an AmP involves formation of several oligomers, protofibrils, and fibrils as summarized in Figure 1. Here, the species placed in the rectangle stand for the soluble oligomers. Conversion of large assemblies such as protofibrils and fibrils into smaller ones is slow, and formation of the large assemblies can be considered as irreversible or partially irreversible. As a new therapeutic option for amyloidoses, we have proposed reduction of the level of the pathogenic oligomers of AmPs through peptide cleavage. As shown in Figure 1 where the cleavage agent is indicated as (R)-(LCo), cleavage of an AmP included in a target oligomer reduces the concentration of the target oligomer, leading to decreases in the concentrations of other oligomers which readily transform into the target oligomer. The successful discovery of cleavage agents for oligomers of Aβ42 and h-IAPP suggested that new therapeutic methods for amyloidoses may be obtained by using artificial proteases. In the present study, we have tested whether artificial proteases can be also designed for the oligomers of αsynuclein which is implicated as the pathogenic species of Parkinson’s disease. A slightly modified form (MW 15500) of α-synuclein was obtained in the present study as described in the Experimental Section. To examine the degree of aggregation of αsynuclein, a filtration experiment was performed: A solution (70 μM) of α-synuclein was shaken (600 rpm) at 37 °C and pH 7.50 (50 mM HEPES). At various time intervals, an aliquot (20 μL) of the mixture was passed through a membrane with the pore size of 0.22 μm (Millipore MillexGV 4MM). The amount of α-synuclein that passed through the filter was measured according to the procedure described previously. Results of the filtration experiment are summarized in Figure 2. Because the pore of the filter was fairly large, the fraction blocked by the filter represents large protofilbrils and fibrils as well as aggregates adsorbed onto the reaction vessel (Eppendorf tube). The results reveal that a major portion of α-synuclein remains as aggregates that can pass through the filter with a pore size of 0.22 μm within several days. The four cleavage agents (A-D) reported to cleave soluble oligomers of Aβ42 and h-IAPP were tested with αsynuclein. In A-D, the Co(III) complex of cyclen (Co(III)cyclen) is exploited as the catalytic site for peptide cleavage and the organic moieties are employed as binding site to recognize the target oligomers of AmPs. A solution of αsynuclein (70 μM) was incubated with one of A-D at 37 °C for various periods and cleavage of α-synuclein was examined by MALDI-TOF MS. Cleavage agents A and B exhibited cleavage activity whereas C and D manifested no activity. The product solution obtained by the reaction of α-