Nucleation of Alpha-Synuclein Aggregation in Live Cells by Quantum Dots

. ELIZABETH JARES-ERIMAN., M. JULIA ROBERTI, THOMAS M. JOVIN

Boston, MA, EEUU

Congreso; Material Research Society Meeting; 2007

Material Research Society Meeting

We exploited the multivalency of streptavidin-conjugate QDs to assemble artificial nucleation centers that exert a biological impact in a cellular event of our interest, namely the aggregation of α-synuclein. α-synuclein is an abundant and natively unfolded pre-synaptic protein (14 kDa) related to the pathogenesis of Parkinson’s disease, where aberrant, amyloid-like deposits of this protein are characteristic. There is growing evidence to the cytotoxicity of α-synuclein oligomerization for motor neurons, and many efforts are devoted to unveil the mechanisms underlying α-synuclein physiological function and pathological self-association. In a previous work, we fused α-synuclein with a 12-aa peptide bearing a tetracysteine tag (CCPGCC) for binding fluorogenic biarsenical compounds (e.g. FlAsH, ReAsH). Extensive characterization of the recombinant protein (α-synuclein-C4) stated that the biophysical, biochemical and aggregation properties matched those of the wild-type protein, in vitro and inside cells. We will show that α-synuclein-conjugated QDs can act as nucleation seeds that induce aggregation of FlAsH-labeled α-synuclein-C4 (α-synuclein-C4-FlAsH). We were able to track simultaneously the fate of both the nucleation seeds and the bulk protein through fluorescence spectroscopy and microscopy in vitro and inside cells and proposed a nucleation-propagation mechanism for α-synuclein fibrillation. We evaluated the ability of QDs as actuators for α-synuclein aggregation from two aspects: their effective concentration in the aggregation mixture, related to the occurrence of nucleation points in a typical aggregation assay, and a modulation upshot from the different degrees of α-synuclein-B coverage attainable by incubation with different QDs:α-synuclein-B ratios.