Spontaneous Isomerization of Long-Lived Proteins Provides a Molecular Mechanism for the Lysosomal Failure Observed in Alzheimer’s Disease

Abstract

Proteinaceous aggregation is a well-known observable in Alzheimer’s disease (AD), but failure and storage of lysosomal bodies within neurons is equally ubiquitous and actually precedes bulk accumulation of extracellular amyloid plaque. In fact, AD shares many similarities with certain lysosomal storage disorders though establishing a biochemical connection has proven difficult. Herein, we demonstrate that isomerization and epimerization, which are spontaneous chemical modifications that occur in long-lived proteins, prevent digestion by the proteases in the lysosome (namely, the cathepsins). For example, isomerization of aspartic acid into l -isoAsp prevents digestion of the N-terminal portion of Aβ by cathepsin L, one of the most aggressive lysosomal proteases. Similar results were obtained after examination of various target peptides with a full series of cathepsins, including endo-, amino-, and carboxy-peptidases. In all cases peptide fragments too long for transporter recognition or release from the lysosome persisted after treatment, providing a mechanism for eventual lysosomal storage and bridging the gap between AD and lysosomal storage disorders. Additional experiments with microglial cells confirmed that isomerization disrupts proteolysis in active lysosomes. These results are easily rationalized in terms of protease active sites, which are engineered to precisely orient the peptide backbone and cannot accommodate the backbone shift caused by isoaspartic acid or side chain dislocation resulting from epimerization. Although Aβ is known to be isomerized and epimerized in plaques present in AD brains, we further establish that the rates of modification for aspartic acid in positions 1 and 7 are fast and could accrue prior to plaque formation. Spontaneous chemistry can therefore provide modified substrates capable of inducing gradual lysosomal failure, which may play an important role in the cascade of events leading to the disrupted proteostasis, amyloid formation, and tauopathies associated with AD.

Spontaneous Isomerization of Asp387 in Tau is Diagnostic for Alzheimer’s Disease: An Endogenous Indicator of Reduced Autophagic Flux

“…Furthermore, once autophagic flux slows sufficiently to allow isomerization, a destructive cycle is enabled because isomerization itself is refractory to and interferes with autophagic degradation. 25…”

“…Although the isomerization rate of Asp387 in tau is not known, measured rates of Asp isomerization in other peptides were found to proceed at rates of ~1% per day or less. 25 If isomerization of Asp387 in tau proceeds similarly, then the amount of isomerization reported in Fig. 1 would require up to 60 days (or more) for the most dramatic cases.…”

“…Isomerization of Aβ has been examined primarily within the context of its influence on aggregation, although recent results have illustrated that preventing lysosomal degradation may be more injurious. 25 In the case of tau, isomerization has only been studied in a handful of reports. Initial work relied on methylation of L-isoAsp by protein isoaspartyl methyl-transferase (PIMT) to quantify isomerization of tau at the protein level.…”

Tau Is Truncated in Five Regions of the Normal Adult Human Brain

“…Over time, spontaneous cleavages, in particular at Asp [ 44 ], Asn [ 29 ], and Ser [ 45 ] residues, take place. As to why Tau fragments appear not to be degraded further within neurons (or possibly glial cells), it can be speculated that they remain tightly bound to microtubules or that other common age-related PTMs such as isomerisation and racemisation may impair the ability of the cell’s protein degradation machinery to adequately process these modified polypeptides [ 57 ]. In other human tissues, significant cleavage as well as other PTMs can be found in life-long proteins isolated from people even as early as the second decade of life [ 58 ].…”

A Curcumin Analog Exhibits Multiple Biologic Effects on the Pathogenesis of Alzheimer’s Disease and Improves Behavior, Inflammation, and β-Amyloid Accumulation in a Mouse Model

“…One provocative theory in AD pathogenesis is the role of mTOR and the autophagy machinery mechanism. Recently, Lambeth and colleagues reported that long-lived protein can spontaneously modify substrates and gradually disrupt the lysosomal protein degradation pathway [50]. Therefore, activation of the cellular autophagy lysosomal system regulated by mTOR may provide one alternate strategy for aggregated protein clearance.…”