Application of NanoBiT for Monitoring Dimerization of the Null Hong Kong Variant of α-1-Antitrypsin, NHK, in Living Cells
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In this study, we investigated expression and dimerization of an ER-associated degradation (ERAD) substrate, a null Hong Kong variant of α-1-antitrypsin (NHK) using immunoblotting assay and a novel NanoLuc complementary reporter system called the NanoBiT (NB) assay. This NB-tagged NHK made it possible to monitor the intra- and extracellular status of NHK in living cells. The values for this NB assay fluctuated in response to distinct pharmacological stimuli and co-transfection of several ERAD-related factors. We then focused on mesencephalic astrocyte-derived neurotrophic factor (MANF), an unclarified ATF6/IRE1-downstream target, and established MANF-deficient Neuro2a (N2a) cells using CRISPR/Cas9 system. MANF-deficient N2a significantly elevated OS-9 protein after tunicamycin treatment; however, no specific differences in intra- and extracellular status of NHK protein were observed between wild-type and MANF-deficient cells. Taken together, intrinsic MANF in N2a cells is not strongly associated with the accumulation and clearance of unfolded proteins within the ER under current condition, but this novel NB assay is a useful approach for characterizing the protein status including ERAD substrates.
KeywordsMANF ER stress ERAD NanoBiT UPR
We thank Dr. Nobuko Hosokawa for the critical reading of this manuscript. We are grateful to Dr. Wei Liu and Dr. Jennifer Lippincott-Schwartz, Dr. Maurizio Molinari, Dr. Nobuko Hosokawa, Dr. Kazuhiro Nagata and Promega Corporation for providing the HA-tagged Sar1 (H79G), OS-9 and NHK genes and NB assay system, respectively. This work is, in part, supported by Research Fellowships for Young Scientists (to J.N.), Grant-in-Aid for Challenging Exploratory Research (No. 17K19901 to K.O.), Grant-in-Aid for Challenging Exploratory Research (No. 26670692, to F.A.), and the OGAWA Science and Technology Foundation (to K.O.).
- 7.Haze, K., Yoshida, H., Yanagi, H., Yura, T., & Mori, K. (1999). Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress. Molecular Biology of the Cell, 10, 3787–3799.CrossRefPubMedPubMedCentralGoogle Scholar
- 19.Glembotski, C. C., Thuerauf, D. J., Huang, C., Vekich, J. A., Gottlieb, R. A., & Doroudgar, S. (2012). Mesencephalic astrocyte-derived neurotrophic factor protects the heart from ischemic damage and is selectively secreted upon sarco/endoplasmic reticulum calcium depletion. Journal of Biological Chemistry, 287, 25893–25904.CrossRefPubMedGoogle Scholar
- 22.Lindholm, P., & Saarma, M. (2010). Novel CDNF/MANF family of neurotrophic factors. Devlopmental Neurobiology, 70, 360–371.Google Scholar
- 25.Oh-hashi, K., Hirata, Y., & Kiuchi, K. (2013). Transcriptional regulation of mouse mesencephalic astrocyte-derived neurotrophic factor in Neuro2a cells. Cellular & Molecular Biology Letters, 18, 398–415.Google Scholar
- 43.Horimoto, S., Ninagawa, S., Okada, T., Koba, H., Sugimoto, T., Kamiya, Y., Kato, K., Takeda, S., & Mori, K. (2013). The unfolded protein response transducer ATF6 represents a novel transmembrane-type endoplasmic reticulum-associated degradation substrate requiring both mannose trimming and SEL1L protein. Journal of Biological Chemistry, 288, 31517–31527.CrossRefPubMedGoogle Scholar
- 45.Okada, T., Yoshida, H., Akazawa, R., Negishi, M., & Mori, K. (2002). Distinct roles of activating transcription factor 6 (ATF6) and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) in transcription during the mammalian unfolded protein response. Biochemical Journal, 366, 585–594.CrossRefPubMedPubMedCentralGoogle Scholar