LC–MS Reveals Isomeric Inhibition of Proteolysis by Lysosomal Cathepsins

Pandey, Gaurav; Julian, Ryan R.

doi:10.1002/anse.202200017

Cited by 3 publications

(2 citation statements)

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“…Similarly, peptide neurotransmitters and toxins have been found to contain d -amino acids which increase lifetimes by conferring resistance to proteolysis. , In terms of LLPs, isomerization can potentially disrupt proteostasis by preventing the breakdown into constituent amino acids. For example, studies of the lysosomal cathepsins have shown that isomerized substrates cannot be fully digested, which could eventually lead to lysosomal storage observed in Alzheimer′s disease (AD). , …”

Section: Introductionmentioning

confidence: 99%

“…For example, studies of the lysosomal cathepsins have shown that isomerized substrates cannot be fully digested, which could eventually lead to lysosomal storage observed in Alzheimer′s disease (AD). 15,16 Although the lysosome appears to be unable to degrade isomerized substrates, the ubiquitin−proteasome system is another possible pathway for protein degradation. Within the mammalian cell, the 26S proteasome is a major complex composed of a core 20S unit responsible for enzymatic digestion and one to two outer 19S regulatory units that act together for the digestion and recycling of protein/peptide substrates.…”

Section: ■ Introductionmentioning

confidence: 99%

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Influence of Asp Isomerization on Trypsin and Trypsin-like Proteolysis

et al. 2022

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Long-lived proteins (LLPs), although less common than their short-lived counterparts, are increasingly recognized to play important roles in age-related diseases such as Alzheimer's. In particular, spontaneous chemical modifications can accrue over time that serve as both indicators of and contributors to disrupted autophagy. For example, isomerization in LLPs is common and occurs in the absence of protein turnover while simultaneously interfering with the protein turnover by impeding proteolysis. In addition to the biological implications this creates, isomerization may also interfere with its own analysis. To clarify, bottom-up proteomics experiments rely on protein digestion by proteases, most commonly trypsin, but the extent to which isomerization might interfere with trypsin digestion is unknown. Here, we use a combination of liquid chromatography and mass spectrometry to examine the effect of isomerization on proteolysis by trypsin and chymotrypsin. Isomerized aspartic acid and serine residues (which represent the most common sites of isomerization in LLPs) were placed at various locations relative to the preferred protease cleavage point to evaluate the influence on digestion efficiency. Trypsin was found to be relatively tolerant of isomerization, except when present at the residue immediately C-terminal to Arg/Lys. For chymotrypsin, the influence of isomerization on digestion was less predictable, resulting in long-range interference for some isomer/peptide combinations. Given the trypsin-and chymotrypsin-like behaviors of the 20S proteasome, and to further establish the biological relevance of isomerization in LLPs, substrates with isomerized sites were also tested against proteasomal degradation. Significant disruption of 20S proteolysis was observed, suggesting that if LLPs persist long enough to isomerize, it will be difficult for the cells to digest them.

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Section: Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Influence of Asp Isomerization on Trypsin and Trypsin-like Proteolysis

et al. 2022

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Profiling the Misfolded Proteome in Human Disease

Onwudiwe,

Genereux

2024

Israel Journal of Chemistry

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Changes in protein homeostasis are broadly implicated in many disease states, including amyloidoses, neurodegenerative diseases, cancer, and normal aging. Although this relationship has been fruitful for identifying and developing therapeutic strategies, it is challenging to identify which proteins are misfolding. New technologies have recently emerged that enable proteome‐wide interrogation of protein conformation and stability. In this review, we describe these technologies, and how they have been used to identify proteins whose folding changes between disease states. We discuss some of the challenges in this emerging field, and the potential for misfolded protein profiling to provide insight into human disease.

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Unusually Rapid Isomerization of Aspartic Acid in Tau

Shoff,

Van Orman,

Onwudiwe

et al. 2024

Preprint

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Spontaneous chemical modifications in long-lived proteins can potentially change protein structure in ways that impact proteostasis and cellular health. For example, isomerization of aspartic acid interferes with protein turnover and is anticorrelated with cognitive acuity in Alzheimer's disease. However, few isomerization rates have been determined for Asp residues in intact proteins. To remedy this deficiency, we used protein extracts from SH-SY5Y neuroblastoma cells as a source of a complex, brain-relevant proteome with no baseline isomerization. Cell lysates were aged in vitro to generate isomers, and extracted proteins were analyzed by data-independent acquisition (DIA) liquid chromatography-mass spectrometry (LC-MS). Although no Asp isomers were detected at Day 0, isomerization increased across time and was quantifiable for 105 proteins by Day 50. Data analysis revealed that isomerization rate is influenced by both primary sequence and secondary structure, suggesting that steric hindrance and backbone rigidity modulate isomerization. Additionally, we examined lysates extracted under gentle conditions to preserve protein complexes and found that protein-protein interactions often slow isomerization. Base catalysis was explored as a means to accelerate Asp isomerization due to findings of accelerated asparagine deamidation. However, no substantial rate enhancement was found for isomerization, suggesting fundamental differences in acid-base chemistry. With an enhanced understanding of Asp isomerization in proteins in general, we next sought to better understand Asp isomerization in tau. In vitro aging of monomeric and aggregated recombinant tau revealed that tau isomerizes significantly faster than any similar protein within our dataset, which is likely related to its correlation with cognition in Alzheimer's disease.

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LC–MS Reveals Isomeric Inhibition of Proteolysis by Lysosomal Cathepsins

Cited by 3 publications

References 55 publications

Influence of Asp Isomerization on Trypsin and Trypsin-like Proteolysis

Influence of Asp Isomerization on Trypsin and Trypsin-like Proteolysis

Profiling the Misfolded Proteome in Human Disease

Unusually Rapid Isomerization of Aspartic Acid in Tau

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