Lysosomal malfunction is associated with many age-related disorders including Alzheimer’s disease and macular degeneration. The lysosome plays a crucial role in autophagy and is responsible for breaking down proteins into amino acids, but certain spontaneous chemical modifications associated with long-lived proteins can interfere with this process. In particular, isomerization/epimerization (which can accumulate rapidly in peptides or disordered protein domains containing aspartic acid or by deamidation of asparagine) are known to interfere with proteolysis. Unfortunately, isomerization/epimerization do not lead to a change in mass, making them invisible to traditional proteomics. However, it is possible to distinguish peptide isomers in MS/MS spectra if the dissociation method is sensitive to structural differences. Typically collision-induced dissociation struggles to identify isomers, but we have demonstrated that enhanced structural sensitivity of radical-directed dissociation is ideal for isomer/epimer identification. We have identified many sites of isomerization in long-lived proteins derived from the eye lens, but have also demonstrated that such modifications can occur in a few weeks and are relevant to other tissues.
Here, we explore the effect of isomerization/epimerization on lysosomal degradation by examining proteolysis of a variety of model peptides with a suite of cathepsins, the major proteases in the lysosome. It is demonstrated that proteolysis is prevented to varying degrees by all isomer/epimers. In some instances, protection of 13 residues is afforded by a single, centrally located isomerized residue. This behavior can easily be rationalized by examining the structural details of protease active sites, which are defined by a binding groove where the peptide backbone must be precisely localized for hydrolysis. Isomerization/epimerization both represent structural changes that cannot be accommodated by these binding grooves.
These results foreshadow consequences for autophagy and proteostasis. The inability of lysosomes to break down isomerized peptides should lead to eventual lysosomal storage, which is observed in Alzheimer’s disease.