Abstract
Endoplasmic reticulum (ER) stress induced by disruption of protein folding activates the unfolded protein response (UPR), which while generally pro-survival in effect can also induce cell death under severe ER stress. 24(S)-hydroxycholesterol (24S-OHC), which is enzymatically produced in the ER of neurons, plays an important role in maintaining brain cholesterol homeostasis but also shows neurotoxicity when subjected to esterification by acyl-CoA:cholesterol acyltransferase 1 (ACAT1) in the ER. In this study, we demonstrated that the accumulation of 24S-OHC esters in human neuroblastoma SH-SY5Y cells evoked the UPR with substantially no pro-survival adaptive response but with significant activation of pro-death UPR signaling via regulated IRE1-dependent decay (RIDD). We further found that accumulation of 24S-OHC esters caused disruption of ER membrane integrity and release of ER luminal proteins into cytosol. We also found that de novo synthesis of global proteins was robustly suppressed in 24S-OHC-treated cells. Collectively, these results show that ER dysfunction and the accompanying RIDD-mediated pro-death UPR signaling and global protein synthesis inhibition are responsible for 24S-OHC ester-induced unconventional cell death.
Highlights
Upon accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER)—a condition referred to as ER stress—cells activate an adaptive response—this response being referred to as the unfolded protein response (UPR)—to restore ER homeostasis and maintain the fidelity of protein-folding[1]
We found that esterification of 24S-OHC evoked the UPR but the downstream pro-survival adaptive response was not substantially activated, finding instead that regulated IRE1dependent decay (RIDD) is implicated in 24S-OHC-induced cell death
The same shift in the IRE1α band was observed in cells treated with 50 μM 24S-OHC for 6 h, suggesting that 24S-OHC treatment induced IRE1α phosphorylation. 24S-OHC-induced IRE1α phosphorylation was inhibited by cotreatment with F12511 but not by Nec-1 (Fig. 1a)
Summary
Upon accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER)—a condition referred to as ER stress—cells activate an adaptive response—this response being referred to as the unfolded protein response (UPR)—to restore ER homeostasis and maintain the fidelity of protein-folding[1]. The UPR comprises three major signaling pathways, which are respectively mediated by inositol-requiring enzyme 1 (IRE1), protein kinase RNA-like ER kinase (PERK), and activating transcription factor 6 (ATF6). XBP1s induces the expression of several genes involved in the UPR to activate pro-survival mechanisms. IRE1 RNase activity contributes to posttranscriptional degradation of a subset of ER-localized mRNAs through a process known as regulated IRE1dependent decay (RIDD)[7,8,9]. RIDD may help to reduce the folding load of nascent proteins and alleviate ER stress as a pro-survival mechanism, continuous decay of RIDD substrates by unmitigated ER stress leads to prodeath outputs[4,10]. Activated IRE1 promotes apoptosis through the activation of apoptosis signal-regulated kinase 1 (ASK1; known as MAP3K5), JUN N-
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