UCLA

Alterations in protein stability and propensity for aggregation are hallmarks of many prevalent and costly age-related diseases, and are actively investigated in cases like Alzheimer’s and cataracts. However, as expensive clinical trials of drugs based on the native targets fail, we must reconsider what the toxic species are within these protein aggregation diseases. One hypothesis focuses on the alteration of aggregative properties by spontaneous protein modifications, which may generate more toxic polymorphs. One such age-related post-translational modification, the isomerization of aspartate residues (L-isoaspartate, L-isoAsp), is a pervasive modification where preliminary evidence for such aggregation enhancement has been obtained. The primary goal of this thesis has been to characterize how this form of protein damage is linked to aggregation of proteins associated with specific aging diseases, as well as how this modification is maintained in the functioning cell. In the context of the water-soluble extracts of aging mammalian lens, this work has shown that L-isoAsp accumulates to high levels, primarily within aggregated low molecular weight species. In vitro experiments based on our localization of L-isoAsp in the lens revealed that introduction of the L-isoAsp modification in a water-soluble derived peptide was accompanied by decreased aggregative properties, while the opposite result was observed for a water-insoluble derived peptide. In further experiments with segments of the Alzheimer’s disease-associated amyloid-β (Aβ) peptide it was demonstrated that the rate of Aβ fibril formation is greatly accelerated with an isoaspartyl residue in agreement with earlier literature. Structural studies revealed the L-isoAsp residue facilitates the formation of a novel protofilament interface, which may represent a distinct polymorph of the full-length Aβ peptide. Here we also begin the characterization of a novel L-isoAsp maintenance pathway distinct from the canonical L-isoAsp repair enzyme – the protein-L-isoaspartyl (D-aspartyl) O-methyltransferase or protein carboxyl methyltransferase (PCMT1) - through the protein carboxyl domain-containing proteins (PCMTDs). We have shown that these proteins assemble with cullin-RING ligase proteins and lower detectable levels of L-isoAsp in vitro. From this work, it is clear that L-isoAsp residues have significant consequences in protein aggregation diseases, and are attenuated in cells through multiple pathways.