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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 ?-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.

SUBMITTER: Dasgupta S

PROVIDER: S-EPMC4117522 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Proteasome inhibitors alter levels of intracellular peptides in HEK293T and SH-SY5Y cells.

Dasgupta Sayani S Castro Leandro M LM Dulman Russell R Yang Ciyu C Schmidt Marion M Ferro Emer S ES Fricker Lloyd D LD

PloS one 20140731 7

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 us ...[more]

PMID: 25079948

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Project description:Inositol-polyphosphate-induced Ca2+ mobilization was investigated in saponin-permeabilized SH-SY5Y human neuroblastoma cells. Ins(1,4,5)P3 induced a dose-related release from intracellular Ca2+ stores with an EC50 (concn. giving half-maximal effect) of 0.1 microM and a maximal release of 70%. Ins(1,3,4)P3, DL-Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5 did not evoke Ca2+ mobilization in these cells when used at concentrations up to 10 microM. However, Ins(1,3,4,5)P4 was found to release Ca2+ in a dose-related manner, but the response was dependent on the source of Ins(1,3,4,5)P4 used. When commercially available D-Ins(1,3,4,5)P4 was used, the EC50 and maximal response values were 1 microM and 50% respectively, compared with values for chemically synthesized DL-Ins(1,3,4,5)P4 of 2 microM and 25%. The enhanced maximal response of commercial D-Ins(1,3,4,5)P4 was decreased by pretreatment with rat brain crude Ins(1,4,5)P3 3-kinase and was therefore concluded to be indicative of initial Ins(1,4,5)P3 contamination of the Ins(1,3,4,5)P4 preparation. When metabolism of DL-Ins(1,3,4,5)P4 (10 microM) in these cells at 25 degrees C was investigated by h.p.l.c., substantial amounts of Ins(1,4,5)P3 (0.2 microM) and Ins(1,3,4)P3 (0.8 microM) were found to be produced within 3 min. Analysis of DL-Ins(1,3,4,5)P4 incubation with cells at 4 degrees C, however, indicated that metabolism had been arrested ([3H]Ins(1,4,5)P3 detection limits were estimated to be approx. 0.01 microM). When chemically synthesized DL-Ins(1,3,4,5)P4 and incubation conditions of low temperature were used, the Ca2(+)-releasing properties of this compound were established to be 1 microM and 19% for the EC50 and maximal response values respectively. The results obtained strongly suggest that Ins(1,3,4,5)P4 alone has the ability to release intracellular Ca2+. However, in the presence of sub-maximal concentrations of Ins(1,4,5)P3, Ca2+ release appears to be synergistic with Ins(1,3,4,5)P4, but at supramaximal concentrations not even additive effects are observed.

Dataset Information

Proteasome inhibitors alter levels of intracellular peptides in HEK293T and SH-SY5Y cells.

Publications

Proteasome inhibitors alter levels of intracellular peptides in HEK293T and SH-SY5Y cells.

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