Abstract

The proteasome cleaves intracellular proteins into peptides. Earlier studies found that treatment of human embryonic kidney 293T (HEK293T) cells with epoxomicin (an irreversible proteasome inhibitor) generally caused a decrease in levels of intracellular peptides. However, bortezomib (an antitumor drug and proteasome inhibitor) caused an unexpected increase in the levels of most intracellular peptides in HEK293T and SH-SY5Y cells. To address this apparent paradox, quantitative peptidomics was used to study the effect of a variety of other proteasome inhibitors on peptide levels in HEK293T and SH-SY5Y cells. Inhibitors tested included carfilzomib, MG132, MG262, MLN2238, AM114, and clasto-Lactacystin β-lactone. Only MG262 caused a substantial elevation in peptide levels that was comparable to the effect of bortezomib, although carfilzomib and MLN2238 elevated the levels of some peptides. To explore off-target effects, the proteosome inhibitors were tested with various cellular peptidases. Bortezomib did not inhibit tripeptidyl peptidase 2 and only weakly inhibited cellular aminopeptidase activity, as did some of the other proteasome inhibitors. However, potent inhibitors of tripeptidyl peptidase 2 (butabindide) and cellular aminopeptidases (bestatin) did not substantially alter the peptidome, indicating that the increase in peptide levels due to proteasome inhibitors is not a result of peptidase inhibition. Although we cannot exclude other possibilities, we presume that the paradoxical increase in peptide levels upon treatment with bortezomib and other inhibitors is the result of allosteric effects of these compounds on the proteasome. Because intracellular peptides are likely to be functional, it is possible that some of the physiologic effects of bortezomib and carfilzomib arise from the perturbation of peptide levels inside the cell.

Highlights

  • A major pathway of intracellular protein degradation involves the proteasome, a multi-subunit enzyme complex that resides in the cytosol and nucleus [1,2]

  • Many proteasome degradation products are rapidly destroyed by aminopeptidases [11], mass spectrometry based peptidomic studies detected a large number of protein-derived peptides in animal tissues and cell lines [12,13]

  • Proteins are converted into peptides by the proteasome, and it was generally assumed that the resulting peptides were rapidly degraded into amino acids by cellular peptidases [6,11]

Read more

Summary

Introduction

A major pathway of intracellular protein degradation involves the proteasome, a multi-subunit enzyme complex that resides in the cytosol and nucleus [1,2]. Proteins destined for degradation, usually by the covalent addition of ubiquitin, are transported into the interior of the proteasome where they encounter the active protease subunits. A small percentage of the peptides produced by the proteasome are transported into the endoplasmic reticulum and incorporated into major histocompatibility complex (MHC) class I proteins, which present the peptides on the cell surface [10]. Many proteasome degradation products are rapidly destroyed by aminopeptidases [11], mass spectrometry based peptidomic studies detected a large number of protein-derived peptides in animal tissues and cell lines [12,13]. Several studies have found that intracellular peptides are functional and influence signal transduction as well as other cellular processes [14,15,16,17]

Methods
Results
Conclusion
Full Text
Paper version not known

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call