Engineering of alpha synuclein to create aggregation-defective variants, aggregation modulators and molecular probes.

摘要

The objective of this work was to engineer protein variants of alpha synuclein (αS) in order to create protein variants for the modulation and detection of αS aggregation, implicated in the pathology of Parkinson's disease (PD). By engineering the linker region of αS, which connects β strand forming domains critical for aggregation, it is possible to modulate the aggregative properties of αS, while maintaining all physicochemical properties of the native sequence. I identified a novel molecular factor associated with the linker region of αS, namely a propensity to form parallel in-register β sheets, critical for αS aggregation. In addition, incubation of an engineered αS linker variant was able to inhibit αS aggregation. Furthermore, I was able to exploit the self-assembly nature of aggregation in conjunction with the conformation-sensitive biarsenic fluorescent dye, FlAsH, in order to create a molecular probe capable of rapid, specific and quantitative detection of αS oligomeric intermediate species, the potentially cytotoxic species in PD. Collectively, my results highlight the importance of the linker region within the context of αS aggregation and help elucidate a novel therapeutic and diagnostic approach through engineering of the αS linker region to create aggregation inhibitors and aggregate detection systems.