![]() B, real-time qPCR analysis of ATF6 mRNA in SH-SY5Y cells transduced with vectors encoding shRNA against ATF6 or control and treated with 3 μ m Tm or DMSO for 24 h. A, ATF6 protein levels were analyzed by Western blotting using ATF6 antibody. SH-SY5Y cells were transduced with vectors encoding shRNA against ATF6 or scrambled control ( scram). Collectively, our results indicate that from the moment of activation, IRE1 signaling during ER stress has an ATF6-dependent "off-switch."ĪPY29 ATF6 GRP78 IRE1 UPR reporters X-box binding protein 1 (XBP1) c-Jun N-terminal kinase (JNK) cell death endoplasmic reticulum stress (ER stress) fluorescent reporter high content imaging imaging lentivirus protein misfolding stress response unfolded protein response (UPR). Furthermore, inhibition of IRE1 kinase activity or of downstream JNK activity prevented an increase in IRE1 levels during ER stress, suggesting that IRE1 transcription is regulated through a positive feed-forward loop. Moreover, overexpression of the transcriptionally active N-terminal domain of ATF6 reversed the increases in IRE1 levels. Transient increases in both IRE1 mRNA and IRE1 protein levels were observed in response to ER stress, suggesting that IRE1 up-regulation is a general feature of ER stress signaling and was further increased in cells lacking ATF6 expression. We observed that loss of ATF6 expression results in uncontrolled IRE1-reporter activity and increases X box-binding protein 1 ( XBP1) splicing. To investigate cross-talk between these different UPR enzymes, here we developed a high-content live cell screening platform to image fluorescent UPR-reporter cell lines derived from human SH-SY5Y neuroblastoma cells in which different ER stress signaling proteins were silenced through lentivirus-delivered shRNA constructs. When this restoration fails, however, cells undergo apoptosis. These proteins initiate a signaling and transcriptional network termed the unfolded protein response (UPR), which re-establishes cellular proteostasis. These results indicate that a general principle in maintaining membrane tethering is multi-modular self-association.In response to an accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen, three ER transmembrane signaling proteins, inositol-requiring enzyme 1 (IRE1), PRKR-like ER kinase (PERK), and activating transcription factor 6α (ATF6α), are activated. The density of Climp63, likely reflecting the strength of cis interactions, influenced the ER width, which was maintained by trans interactions. The leading helix of the LD was dispensable for homotypic interactions but packing of the 5HB regulated self-association. Charge-based trans associations were formed between the tip of the 5HB and the C-terminus of the LD, consistent with generating a width of ∼50 nm for ER sheets. The LD is highly α-helical, with a flexible leading helix followed by a five-helix bundle (5HB). Here, we analyzed the homotypic interactions of the Climp63 LD using deep learning-predicted structures. However, the molecular basis of luminal spacing remains elusive. Self-association of the Climp63 luminal domain (LD), even though moderate, plays a key role in shaping ER sheets. The width of cisternal structures in the endoplasmic reticulum (ER) is maintained by the ER-resident protein Climp63 (also known as CKAP4).
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