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Molecular characterization of the ER stress-inducible factor CRELD2

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Abstract

Previously, we found by constructing various luciferase reporters that a well-conserved ATF6-binding element in the CRELD2 promoter is activated by transient ATF6 overexpression. In this study, we established ATF6-deficient and ATF4-deficient cell lines to analyze CRELD2 mRNA and protein expression together with that of other ER stress-inducible factors. Our results showed that ATF6 deficiency markedly suppressed tunicamycin (Tm)-induced expression of unglycosylated CRELD2. This reduction reflected a decrease in the CRELD2 transcription level. On the other hand, a putative ATF4-binding site in the mouse CRELD2 promoter did not respond to Tm stimulation, but ATF4 loss resulted in reductions in CRELD2 mRNA and protein expression, accompanied by a decrease in Tm-induced ATF6 expression. In contrast, transient suppression of GADD34, an ATF4 downstream factor, suppressed Tm-induced CRELD2 protein expression without a decrease in ATF6 protein expression. Furthermore, we investigated the association of CRELD2 with a well-known ERAD substrate, namely, an α1-antitripsin truncation mutant, NHK, by generating various CRELD2 and NHK constructs. Coimmunoprecipitation of these proteins was observed only when the cysteine in the CXXC motif on the N-terminal side of CRELD2 was replaced with alanine, and the interaction between the two was found to be disulfide bond-independent. Taken together, these findings indicate that CRELD2 expression is regulated by multiple factors via transcriptional and posttranscriptional mechanisms. In addition, the N-terminal structure of CRELD2, including the CXXC motif, was suggested to play a role in the association of the target proteins. In the future, the identification and characterization of factors interacting with CRELD2 will be useful for understanding protein homeostasis under various ER stress conditions.

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Abbreviations

ATF4:

activating transcription factor 4

ATF6:

activating transcription factor 6

CREB3:

cAMP response element binding protein 3

CREB3L2:

cAMP response element binding protein 3-like 2

CRELD2:

cysteine-rich with EGF-like domains 2

CRISPR/Cas9:

clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins 9

ER:

endoplasmic reticulum

ERAD:

ER-associated degradation

ERSE:

ER stress response element

GADD34:

growth arrest and DNA-damage-inducible protein 34

GADD153:

growth arrest and DNA-damage-inducible protein 153

G3PDH:

glyceraldehyde 3-phosphate dehydrogenase

GRP78:

78 kDa glucose-regulated protein

GRP94:

94 kDa glucose-regulated protein

Herp:

homocysteine-induced ER protein

RT-PCR:

reverse transcription polymerase chain reaction

SEL1L:

suppressor/enhancer of lin-12-like

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Acknowledgements

This study was partially supported by the OGAWA Science and Technology Foundation, Koshiyama Science and Technology Foundation and Grants-in-aid from the Japan Society for the Promotion of Science (JSPS, Japan, KAKENHI, Nos. 19H04030 and 20K21751 to K.O.). This work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia (Grant No. GrantA112 to M.K.). We are grateful to Dr. George Church and Dr. Nobuko Hosokawa for providing the hCas9 and human NHK genes, respectively.

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Authors and Affiliations

  1. Graduate School of Natural Science and Technology, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Shohei Hinaga & Kentaro Oh-hashi

  2. Department of Biomedical Sciences, College of Veterinary Medicine, King Faisal University, 31982, Al-Ahsa, Saudi Arabia

    Mahmoud Kandeel

  3. Department of Pharmacology, Faculty of Veterinary Medicine, Kafrelsheikh University, 33516, Kafrelsheikh, Egypt

    Mahmoud Kandeel

  4. Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Kentaro Oh-hashi

  5. United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Kentaro Oh-hashi

  6. Center for One Medicine Innovative Translational Research (COMIT), Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Kentaro Oh-hashi

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  1. Shohei Hinaga
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S.H. and K.O. performed the experiments; S.H. and K.O. confirmed the results; and M.K. and K.O. designed and prepared the manuscript.

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Correspondence to Kentaro Oh-hashi.

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Hinaga, S., Kandeel, M. & Oh-hashi, K. Molecular characterization of the ER stress-inducible factor CRELD2. Cell Biochem Biophys (2024). https://doi.org/10.1007/s12013-024-01300-1

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