Application of NanoBiT for Monitoring Dimerization of the Null Hong Kong Variant of α-1-Antitrypsin, NHK, in Living Cells

Overview

Journal Mol Biotechnol

Publisher Springer

Specialties Biotechnology
Molecular Biology

Date 2018 Jun 8

PMID 29876740

Citations 4

Authors

Junpei Norisada

Keito Fujimura

Fumimasa Amaya

Hiroki Kohno

Yoko Hirata

Kentaro Oh-Hashi

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

Molecular characterization of the ER stress-inducible factor CRELD2.

Hinaga S, Kandeel M, Oh-Hashi K Cell Biochem Biophys. 2024; 82(2):1463-1475.

PMID: 38753249 DOI: 10.1007/s12013-024-01300-1.

Lumit: A Homogeneous Bioluminescent Immunoassay for Detecting Diverse Analytes and Intracellular Protein Targets.

Brian Hwang B, Alves J, Lazar D, Nath N, Engel L, OBrien M Methods Mol Biol. 2023; 2612:195-224.

PMID: 36795369 DOI: 10.1007/978-1-0716-2903-1_15.

A homogeneous bioluminescent immunoassay to probe cellular signaling pathway regulation.

Brian Hwang B, Engel L, Goueli S, Zegzouti H Commun Biol. 2020; 3:8.

PMID: 31909200 PMC: 6941952. DOI: 10.1038/s42003-019-0723-9.

Characterization of IRE1α in Neuro2a cells by pharmacological and CRISPR/Cas9 approaches.

Oh-Hashi K, Kohno H, Kandeel M, Hirata Y Mol Cell Biochem. 2019; 465(1-2):53-64.

PMID: 31834612 DOI: 10.1007/s11010-019-03666-w.

References

Apostolou A, Shen Y, Liang Y, Luo J, Fang S . Armet, a UPR-upregulated protein, inhibits cell proliferation and ER stress-induced cell death. Exp Cell Res. 2008; 314(13):2454-67. PMC: 6719340. DOI: 10.1016/j.yexcr.2008.05.001. View

Oh-Hashi K, Hirata Y, Kiuchi K . Transcriptional regulation of mouse mesencephalic astrocyte-derived neurotrophic factor in Neuro2a cells. Cell Mol Biol Lett. 2013; 18(3):398-415. PMC: 6275609. DOI: 10.2478/s11658-013-0096-x. View

Ellgaard L, Helenius A . Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol. 2003; 4(3):181-91. DOI: 10.1038/nrm1052. View

Kny M, Standera S, Hartmann-Petersen R, Kloetzel P, Seeger M . Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like domain-dependent manner. J Biol Chem. 2010; 286(7):5151-6. PMC: 3037627. DOI: 10.1074/jbc.M110.134551. View

Shridhar R, Shridhar V, Rivard S, Siegfried J, PIETRASZKIEWICZ H, Ensley J . Mutations in the arginine-rich protein gene, in lung, breast, and prostate cancers, and in squamous cell carcinoma of the head and neck. Cancer Res. 1996; 56(24):5576-8. View

Fu L, Sztul E . Traffic-independent function of the Sar1p/COPII machinery in proteasomal sorting of the cystic fibrosis transmembrane conductance regulator. J Cell Biol. 2003; 160(2):157-63. PMC: 2172646. DOI: 10.1083/jcb.200210086. View

Dixon A, Schwinn M, Hall M, Zimmerman K, Otto P, Lubben T . NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem Biol. 2015; 11(2):400-8. DOI: 10.1021/acschembio.5b00753. View

Harding H, Zhang Y, Ron D . Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature. 1999; 397(6716):271-4. DOI: 10.1038/16729. View

Sai X, Kokame K, Shiraishi H, Kawamura Y, Miyata T, Yanagisawa K . The ubiquitin-like domain of Herp is involved in Herp degradation, but not necessary for its enhancement of amyloid beta-protein generation. FEBS Lett. 2003; 553(1-2):151-6. DOI: 10.1016/s0014-5793(03)01009-3. View

10.

Ushioda R, Hoseki J, Araki K, Jansen G, Thomas D, Nagata K . ERdj5 is required as a disulfide reductase for degradation of misfolded proteins in the ER. Science. 2008; 321(5888):569-72. DOI: 10.1126/science.1159293. View

11.

Okuda-Shimizu Y, Hendershot L . Characterization of an ERAD pathway for nonglycosylated BiP substrates, which require Herp. Mol Cell. 2007; 28(4):544-54. PMC: 2149893. DOI: 10.1016/j.molcel.2007.09.012. View

12.

Oh-Hashi K, Sugiura N, Amaya F, Isobe K, Hirata Y . Functional validation of ATF4 and GADD34 in Neuro2a cells by CRISPR/Cas9-mediated genome editing. Mol Cell Biochem. 2017; 440(1-2):65-75. DOI: 10.1007/s11010-017-3156-0. View

13.

Zhong X, Shen Y, Ballar P, Apostolou A, Agami R, Fang S . AAA ATPase p97/valosin-containing protein interacts with gp78, a ubiquitin ligase for endoplasmic reticulum-associated degradation. J Biol Chem. 2004; 279(44):45676-84. DOI: 10.1074/jbc.M409034200. View

14.

Hosokawa N, Wada I, Hasegawa K, Yorihuzi T, Tremblay L, HERSCOVICS A . A novel ER alpha-mannosidase-like protein accelerates ER-associated degradation. EMBO Rep. 2001; 2(5):415-22. PMC: 1083879. DOI: 10.1093/embo-reports/kve084. View

15.

Schroder M, Kaufman R . ER stress and the unfolded protein response. Mutat Res. 2004; 569(1-2):29-63. DOI: 10.1016/j.mrfmmm.2004.06.056. View

16.

Norisada J, Hirata Y, Amaya F, Kiuchi K, Oh-Hashi K . A Comparative Analysis of the Molecular Features of MANF and CDNF. PLoS One. 2016; 11(1):e0146923. PMC: 4731063. DOI: 10.1371/journal.pone.0146923. View

17.

Sugimoto T, Ninagawa S, Yamano S, Ishikawa T, Okada T, Takeda S . SEL1L-dependent Substrates Require Derlin2/3 and Herp1/2 for Endoplasmic Reticulum-associated Degradation. Cell Struct Funct. 2017; 42(2):81-94. DOI: 10.1247/csf.17007. View

18.

Hosokawa N, Wada I, Nagasawa K, Moriyama T, Okawa K, Nagata K . Human XTP3-B forms an endoplasmic reticulum quality control scaffold with the HRD1-SEL1L ubiquitin ligase complex and BiP. J Biol Chem. 2008; 283(30):20914-24. PMC: 3258950. DOI: 10.1074/jbc.M709336200. View

19.

Zhu C, Johansen F, Prywes R . Interaction of ATF6 and serum response factor. Mol Cell Biol. 1997; 17(9):4957-66. PMC: 232347. DOI: 10.1128/MCB.17.9.4957. View

20.

Lindholm P, Saarma M . Novel CDNF/MANF family of neurotrophic factors. Dev Neurobiol. 2010; 70(5):360-71. DOI: 10.1002/dneu.20760. View