Abstract
We have previously identified a novel endoplasmic reticulum (ER) stress-inducible protein, namely, cysteine-rich with EGF-like domains 2 (CRELD2), which is predominantly regulated by ATF6. However, few studies on intrinsic CRELD2 have been published. In the present study, we elucidated the expression of intrinsic CRELD2 in mouse tissues and ER stress- treated Neuro2a cells. Among nine tissues we tested, CRELD2 protein in the heart and skeletal muscles was negligible. CRELD2 expression in Neuro2a cells was induced at the late phase after treatment with tunicamycin (Tm) compared with rapid induction of growth arrest and DNA damage inducible gene 153 (GADD153). On the other hand, another ER stress inducer, thapsigargin, increased the intrinsic CRELD2 secretion from Neuro2a cells. We furthermore established CRELD2-deficient Neuro2a cells to evaluate their features. In combination with the NanoLuc complementary reporter system, which was designed to detect protein-protein interaction in living cells, CRELD2 interacted with not only CRELD2 itself but also with ER localizing proteins in Neuro2a cells. Finally, we investigated the responsiveness of CRELD2-deficient cells against Tm-treatment and found that CRELD2 deficiency did not affect the expression of genes triggered by three canonical ER stress sensors but rendered Neuro2a cells vulnerable to Tm-stimulation. Taken together, these findings provide the novel molecular features of CRELD2, and its further characterization would give new insights into understanding the ER homeostasis and ER stress-induced cellular dysfunctions.
Highlights
Cysteine-rich with EGF-like domains 2 (CRELD2) was first identified as a protein that interacts with human neuronal nicotinic acetylcholine receptor α4 and β2 subunits by the yeast two-hybrid screening and overexpressing study[1]
cysteine-rich with EGF-like domains 2 (CRELD2) protein was detected at approximately 50 kDa (Fig. 1B), which is almost accorded with the predicted molecular size and CRELD2-overexpressing study[18]
We further evaluated the expression of typical endoplasmic reticulum (ER) stress-inducible factors which are downstream of activating transcription factor 6 (ATF6), PKR-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme 1 (IRE1), respectively
Summary
Cysteine-rich with EGF-like domains 2 (CRELD2) was first identified as a protein that interacts with human neuronal nicotinic acetylcholine receptor α4 and β2 subunits by the yeast two-hybrid screening and overexpressing study[1]. CRELD2 was reported to be one of novel androgen receptor target genes in prostate cancer[2]; its molecular features have not been fully elucidated. Some ER resident molecular chaperones, such as 78 kDa glucose-regulated protein (GRP78), are predominantly regulated by ATF6 and alleviate the stress by properly folding and degrading unfolded proteins inside the ER lumen and membrane[9,10,13,14]. Considering this scenery, CRELD2 expression might be associated with the relief of ER disturbance under certain adverse circumstances. We showed that CRELD2 was not directly associated with typical ER stress-inducible factor expression in response to tunicamycin (Tm), but its depletion rendered the cells vulnerable to Tm stimulation
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