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

Abstract

HeLa cells stably expressing the α chain of T-cell receptor (αTCR), a model substrate of ERAD (ERassociated degradation), 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 ER-associated degradation (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 ofp97/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 ATPase 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.

Differences in endoplasmic-reticulum quality control determine the cellular response to disease-associated mutants of proteolipid protein

“…2B, insets). The periphery of these structures contained both mutant PLPmh and calnexin, resembling the distended ER that has been observed under conditions of chronic ER stress (Alvarez et al, 1999;Dalal et al, 2004;Lass et al, 2008;Lin et al, 1999). The milddisease-associated mutants W162L and G245A were also present A twodimensional model of PLP topology illustrating the four TM domains, cytosolic N-and C-termini, palmitoylation sites (squiggles), and disulphide bonds (straight lines).…”

Structure, biological functions and applications of the AB5 toxins

“…However, treatment with SubAB results in rapid degradation of nearly all cellular BiP in a wide variety of cell lines, effectively mimicking a knock-out phenotype. The toxin has already been used to examine the role of BiP and/or ER stress in ER-associated degradation in HeLa cells 48, and regulation of gap junction function in mesangial cells 49. SubAB has also been employed to demonstrate the critical role of BiP in assembly and egress of cytomegalovirus from infected cells 50 and in production and processing of dengue virus proteins 51.…”

Regulation of Subtilase Cytotoxin-Induced Cell Death by an RNA-Dependent Protein Kinase-Like Endoplasmic Reticulum Kinase-Dependent Proteasome Pathway in HeLa Cells

“…Recently, Xu et al reported that proteasomal degradation of Mcl-1 is required for action of p97/VCP, which mediates disparate cellular functions, including ERAD, via the ubiquitin-proteasome system (51). In HeLa cells, SubAB treatment induced activation of ERAD (20). To address whether ERAD is associated with SubAB-induced apoptosis, we carried out p97/VCP knockdown by siRNA and then determined the effect of SubAB on caspase activation.…”