Decreased ER-associated degradation of α-TCR induced by Grp78 depletion with the SubAB cytotoxin

Agnieszka Lass, Marek Kujawa, Elizabeth McConnell, Adrienne W. Paton, James C. Paton, Cezary Wojcik

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

HeLa cells stably expressing the α chain of T-cell receptor (αTCR), a model substrate of ER-associated degradation (ERAD), were used to analyze the effects of BiP/Grp78 depletion by the SubAB cytotoxin. SubAB induced XBP1 splicing, followed by JNK phosphorylation, eIF2α phosphorylation, upregulation of ATF3/4 and partial ATF6 cleavage. Other markers of ER stress, including elements of ERAD pathway, as well as markers of cytoplasmic stress, were not induced. SubAB treatment decreased absolute levels of αTCR, which was caused by inhibition of protein synthesis. At the same time, the half-life of αTCR was extended almost fourfold from 70 min to 210 min, suggesting that BiP normally facilitates ERAD. Depletion of p97/VCP partially rescued SubAB-induced depletion of αTCR, confirming the role of VCP in ERAD of αTCR. It therefore appears that ERAD of αTCR is driven by at least two different ATP-ase systems located at two sides of the ER membrane, BiP located on the lumenal side, while p97/VCP on the cytoplasmic side. While SubAB altered cell morphology by inducing cytoplasm vacuolization and accumulation of lipid droplets, caspase activation was partial and subsided after prolonged incubation. Expression of CHOP/GADD153 occurred only after prolonged incubation and was not associated with apoptosis.

Original languageEnglish (US)
Pages (from-to)2865-2879
Number of pages15
JournalInternational Journal of Biochemistry and Cell Biology
Volume40
Issue number12
DOIs
StatePublished - 2008
Externally publishedYes

Fingerprint

Cytotoxins
T-Cell Antigen Receptor
Degradation
Phosphorylation
Caspases
HeLa Cells
Half-Life
Cytoplasm
Up-Regulation
Adenosine Triphosphate
Chemical activation
Apoptosis
Membranes
Lipids
Substrates
Proteins

Keywords

  • Cytotoxin
  • endoplasmic reticulum (ER)
  • ER-associated degradation (ERAD)
  • Subtilase
  • T-cell receptor (TCR)
  • Unfolded protein response (UPR)

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

Cite this

Decreased ER-associated degradation of α-TCR induced by Grp78 depletion with the SubAB cytotoxin. / Lass, Agnieszka; Kujawa, Marek; McConnell, Elizabeth; Paton, Adrienne W.; Paton, James C.; Wojcik, Cezary.

In: International Journal of Biochemistry and Cell Biology, Vol. 40, No. 12, 2008, p. 2865-2879.

Research output: Contribution to journalArticle

Lass, Agnieszka ; Kujawa, Marek ; McConnell, Elizabeth ; Paton, Adrienne W. ; Paton, James C. ; Wojcik, Cezary. / Decreased ER-associated degradation of α-TCR induced by Grp78 depletion with the SubAB cytotoxin. In: International Journal of Biochemistry and Cell Biology. 2008 ; Vol. 40, No. 12. pp. 2865-2879.
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AB - HeLa cells stably expressing the α chain of T-cell receptor (αTCR), a model substrate of ER-associated degradation (ERAD), were used to analyze the effects of BiP/Grp78 depletion by the SubAB cytotoxin. SubAB induced XBP1 splicing, followed by JNK phosphorylation, eIF2α phosphorylation, upregulation of ATF3/4 and partial ATF6 cleavage. Other markers of ER stress, including elements of ERAD pathway, as well as markers of cytoplasmic stress, were not induced. SubAB treatment decreased absolute levels of αTCR, which was caused by inhibition of protein synthesis. At the same time, the half-life of αTCR was extended almost fourfold from 70 min to 210 min, suggesting that BiP normally facilitates ERAD. Depletion of p97/VCP partially rescued SubAB-induced depletion of αTCR, confirming the role of VCP in ERAD of αTCR. It therefore appears that ERAD of αTCR is driven by at least two different ATP-ase systems located at two sides of the ER membrane, BiP located on the lumenal side, while p97/VCP on the cytoplasmic side. While SubAB altered cell morphology by inducing cytoplasm vacuolization and accumulation of lipid droplets, caspase activation was partial and subsided after prolonged incubation. Expression of CHOP/GADD153 occurred only after prolonged incubation and was not associated with apoptosis.

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