title: Proteasome inhibitors alter levels of intracellular peptides in HEK293T and SH-SY5Y cells.
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.
authors: Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York, United States of America.
authors: Sayani
authors: S
authors: Dasgupta
authors: Department of Pharmacology, Support Center for Research in Proteolysis and Cell Signaling, Biomedical Sciences Institute, University of São Paulo, São Paulo, SP, Brazil.
authors: Leandro M
authors: LM
authors: Castro
authors: Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York, United States of America.
authors: Russell
authors: R
authors: Dulman
authors: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, United States of America.
authors: Ciyu
authors: C
authors: Yang
authors: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, United States of America.
authors: Marion
authors: M
authors: Schmidt
authors: Department of Pharmacology, Support Center for Research in Proteolysis and Cell Signaling, Biomedical Sciences Institute, University of São Paulo, São Paulo, SP, Brazil.
authors: Emer S
authors: ES
authors: Ferro
authors: Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York, United States of America; Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York, United States of America.
authors: Lloyd D
authors: LD
authors: Fricker
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D001897:Boronic Acids / Q000494:pharmacology*; D000069286:Bortezomib; D045744:Cell Line, Tumor; D057809:HEK293 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology*; D010455:Peptides / Q000378:metabolism*; D061988:Proteasome Inhibitors / Q000494:pharmacology*; D011719:Pyrazines / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D001897:Boronic Acids; D007976:Leupeptins; D009842:Oligopeptides; D010455:Peptides; D061988:Proteasome Inhibitors; D011719:Pyrazines; D000069286:Bortezomib; C524865:carfilzomib; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde