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The active balancing of protein synthesis and degradation, or proteostasis, is an ongoing and critical process in most cells. (1) Proteins must be created, carry out their requisite function, and then be recycled once they are no longer needed or have become nonfunctional. Several pathways are available for protein degradation, including the proteasome, macroautophagy, microautophagy, and chaperone-mediated autophagy. (2,3) The autophagy-related pathways deliver proteins to lysosomes, which are acidic organelles containing a host of hydrolases, including many proteases. (4) Cargo taken into cells via endocytosis is also typically delivered to lysosomes for degradation. Regardless of the pathway, after cargo fuses with a lysosome, endopeptidases cleave proteins at internal sites, shortening proteins to peptides, which are then further digested from both termini by exopeptidases. After protein digestion has been completed, transporter proteins in the lysosomal membrane release (primarily) individual amino acids back into the cytosol for new protein synthesis or energy production. (5) Lysosomes are crucial for maintaining cellular homeostasis, but they are also uniquely susceptible to problems when substrates cannot be hydrolyzed. For example, genetic modifications reducing the efficacy of a lysosomal hydrolase are the most common cause of lysosomal storage disorders. These devastating diseases involve “storage” of failed lysosomal bodies within cells, which eventually leads to cell death and is particularly problematic for postmitotic cells such as neurons. (6) Symptoms in lysosomal storage disorders usually emerge in infancy or childhood, are often associated with neurodegeneration, and are typically fatal. (7)

l - to d - configuration. Peptide isomerization and epimerization do not have readily identifiable bioanalytical signatures, but both modulate structure in a subtle, yet significant, way (see l -isoAsp, protein-isoaspartyl methyl transferase (PIMT), Long-lived proteins (8) are a primary target of the lysosome because they become modified and lose efficacy over time. A well-known example of this occurs with mitophagy, (3) wherein old mitochondria are recycled in their entirety. Contributing factors that lead to long-lived protein deterioration include a variety of spontaneous chemical modifications, i.e., modifications not under enzymatic control. (8) Some of these modifications are very subtle and difficult to detect, including isomerization and epimerization. (9) Isomerization occurs primarily at aspartic acid, when the side chain inserts into and elongates the peptide backbone ( Scheme 1 ). (10) Identical products are also created during deamidation of asparagine, which further results in chemical transformation from one amino acid to another. (11) Epimerization occurs when an amino acid side chain inverts chirality from the- to- configuration. Peptide isomerization and epimerization do not have readily identifiable bioanalytical signatures, but both modulate structure in a subtle, yet significant, way (see Figure 1 ). Studies on the eye lens have shown that epimerization and isomerization are among the most abundant modifications observed in extremely long-lived proteins. (12−14) However, knockout experiments in mice have also revealed the importance of these modifications over much shorter time scales. For example, removal of the repair enzyme for-isoAsp, protein-isoaspartyl methyl transferase (PIMT), (15) leads to lethal accumulation of isomerized protein in just 4–6 weeks. (16,17) This reveals that isomerization of aspartic acid is sufficiently dangerous that an enzyme has evolved to repair it.

Scheme 1 Scheme 1. Pathways for Isomerization of Aspartic Acid and Deamidation of Asparagine

Figure 1 Figure 1. Model structures of the aspartic acid isomers, where the isostructure conformation closest to native backbone orientation is shown. Two views are illustrated for each isomer.

l -only peptides are not biologically active, confirming the importance of the chiral modifications. In addition, it is thought that epimerization is beneficial for these peptides because it allows them to escape, or prolong the time required for, proteolysis. The importance of peptide isomers is further revealed in the uses nature has found for them. For example, single amino acid sites are intentionally epimerized in many venoms and in signaling neuropeptides in crustaceans. (18,19) The corresponding-only peptides are not biologically active, confirming the importance of the chiral modifications. In addition, it is thought that epimerization is beneficial for these peptides because it allows them to escape, or prolong the time required for, proteolysis. (20) In fact, it is well-known that sites of epimerization and isomerization are both generally resistant to protease action, but the ramifications of such chemistry in the context of lysosome function have not been previously examined. Despite this absence, numerous studies have established the importance of protein degradation in lysosomes. For example, knockout mice lacking cathepsin D grow normally for ∼2 weeks but then die before the end of 4 weeks. (21) Examination of the neurons from these mice revealed an abundance of failed lysosomal bodies, similar to those observed in lysosomal storage disorders. Other research has shown that knockout mice lacking cathepsins B and L die within 2–4 weeks of birth. Again, accumulation of failed lysosomal bodies was observed in neurons of these mice. (22) Although cathepsins can also be found outside the lysosome, (23) these results confirm a significant, and likely fatal, impact on the lysosomal system when critical cathepsins are absent.

Amyloid aggregates or proteins that are otherwise insoluble are also targeted to lysosomes for degradation. (24) Amyloid aggregation has also captured the majority of attention as the potential cause of Alzheimer’s disease (AD), but significant evidence also supports lysosomal storage as an underlying cause. For example, AD shares many pathological similarities with lysosomal storage disorders, including prolific storage of failed lysosomal bodies, accumulation of senile plaques, and formation of neurofibrillary tangles. (25,26) In fact, scanning-electron microscopy images of lysosomal storage (in neurons) are virtually indistinguishable between the two diseases. The lysosomal storage observed in AD precedes formation of amyloid deposits, (27) hinting that lysosomal malfunction may occur upstream of the events leading to extracellular amyloid aggregation. The parallels between the two diseases have also been offset by differences. For example, lysosomal storage disorders typically afflict youth and can progress rapidly, while AD typically occurs late in life over a longer time scale. Therefore, a mechanism accounting for the commonalities and differences between the diseases has been difficult to identify, but an intriguing possibility does exist.

modified enzyme or modified transporter to clear waste molecules, failure to digest or transport modified waste molecules would be operative and eventually lead to lysosomal storage. Close examination of another complex age-related disease, macular degeneration, reveals that there is precedence for substrate-induced lysosomal storage. The primary constituents of senile plaques, Aβ and Tau, are both long-lived proteins that are subject to isomerization and epimerization. (8) In fact, Aβ is significantly epimerized and isomerized in the brains of people with AD. (28) If isomerization and epimerization prevent lysosomal protein digestion, then a common link between lysosomal storage disorders and AD would be established. In fact, AD would essentially represent a different type of lysosomal storage disorder, one that operates in reverse of the classical disease. Rather than failure of ato clear waste molecules, failure to digest or transportmolecules would be operative and eventually lead to lysosomal storage. Close examination of another complex age-related disease, macular degeneration, reveals that there is precedence for substrate-induced lysosomal storage. (29)

Herein, we use mass spectrometry (MS) and liquid chromatography (LC) to demonstrate that isomerized or epimerized peptides resist degradation by cathepsins, including both endo- and exopeptidase activity. Important target peptides that are both long-lived and closely associated with AD were examined, including fragments of Aβ and Tau. The results reveal that small peptide fragments composed of residues surrounding isomerized or epimerized sites persist after digestion. Disrupted proteolysis was observed in both isolated reactions on model peptides in full-length Aβ, and in living cells, offering an explanation for the toxicity observed in previous experiments with cell and animal models employing isomerized Aβ (see the discussion below). Additional experiments reveal that the rates of isomerization for the Asp residues in the N-terminal portion of Aβ are fast, providing a pathway for generation of these toxic species that could eventually lead to lysosomal failure and initiate other downstream consequences.