Eukaryotic cells have different mechanisms to degrade damaged proteins. The 203 proteasome, which also .forms the core of the larger, ubiquitin-dependent 26S proteasome, appears to have a major role in clearing the cell of oxidatively damaged proteins. Notably, cellular aging is thought to be accelerated by the accumulation of oxidatively damaged proteins. Assembly of the 20S proteasome from its 14 different subunits is directed by proteasome biogenesis-associated factors 3 and 4 (Pba3/Pba4), which help guide the placement of certain subunits into specific positions;without these chaperones, the outer rings of the 20S proteasome are remodeled into an alternative arrangement. Mutant cells lacking Pba3/Pba4 have a growth advantage under conditions known to cause oxidative damage to proteins.
In Aim 1, the physiological role of the alternative proteasome will be determined. If the growth advantage of the chaperone mutants is primarily due to enhanced degradation of damaged proteins by alternative 20S proteasomes, then the mutant should exhibit faster degradation rates of these proteins. Increased degradation of such proteins in cells making alternative proteasomes could also potentially increase chronological lifespan. This will be assayed by manipulating the levels of alternative relative to standard proteasomes and growing the resulting strains either with or without oxidative stress over extended periods. Understanding how these chaperones direct the subunit arrangement of the 20S proteasome is difficult to study in vivo due to the lack of early assembly intermediates that can be isolated.
Aim 2 proposes an in vitro approach to reconstitute 20S proteasome assembly. This method is amenable to various manipulations that can reveal the Pba3/Pba4 mechanism of action. The composition of the 20S proteasome will be assessed with and without Pba3/Pba4 to determine the underlying method by which these chaperones function.
Many aging-related diseases, such as Alzheimer's and Parkinson's disease, are characterized by the accumulation of oxidatively damaged proteins. The 20S proteasome appears to have a special role in degrading such aberrant protein species. Therefore, understanding how different forms of the 20S proteasome are assembled, which can have a major impact on how efficiently these damaged proteins are degraded, will be essential for preventing and treating these diseases.
