Metabolism of Alpha-Synuclein by the 20S Proteasome

Parkinson's disease is one of several common neurodegenerative disorders that are related by the intracellular aggregation of the neuronal protein alpha-synuclein (alphaSyn) that normally associates with synaptic vesicles. Within the aggregates, a fraction of alphaSyn is truncated at the C-termini, and these truncations are hypothesized to participate in disease pathogenesis. The prevailing model for cytotoxicity of the aggregated protein proposes that oligomeric forms cause dysfunction of the ubiquitin-proteasome pathway of protein degradation, thereby enhancing an alternative pathway that involves ubiquitin-independent degradation by the 20S core particle of the proteasome. The 20S proteasome is known to degrade proteins with regions of intrinsic disorder, which figure prominently in a wide range of neurodegenerative disorders, including alphaSyn in Parkinson's disease. Importantly, some forms of 20S exert an endoproteolytic activity that produces highly amyloidogenic truncations of alphaSyn. An in vitro system containing liposomes and the 20S proteasome was developed to identify the mechanism by which the three point mutations in alphaSyn associated with familial Parkinson's disease exert cytotoxicity. The proteasome produced truncations from all three mutants in the presence of liposomes, but not wildtype alphaSyn. Additionally, the putative cytotoxic oligomer was formed most rapidly in the presence of both 20S proteasome and liposomes. Polyclonal antibodies specific for the C-termini of a set of truncated alphaSyn proteins were successfully developed to detect cleavage products of alphaSyn in vitro and in vivo. To better understand the physiological role of 20S-mediated degradation of intrinsically disordered proteins, the mechanism of recognition and interaction between substrate and enzyme was studied. While the presence of disorder appears to be necessary for 20S degradation, it was found to be insufficient to define a protein as a 20S substrate. Furthermore, a novel correlation was identified between the endoproteolytic activity of 20S and a modification of the alpha6 subunit. The data support a role for the 20S proteasome in Lewy body disease pathogenesis, where it accelerates the formation of cytotoxic species by endoproteolysis of an intrinsically disordered protein. In that regard, a form of the 20S proteasome was identified that may be responsible for the endoproteolytic cleavage of intrinsically disordered proteins in vivo.