Expression analysis and functional characterization of the mouse cysteine-rich with EGF-like domains 2.

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.

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

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.

Application of NanoLuc to monitor the intrinsic promoter activity of GRP78 using the CRISPR/Cas9 system.

In this study, we applied a highly sensitive small luciferase, NanoLuc, to establish a knock-in cell line using the CRISPR/Cas9 system and characterized the endogenous promoter activity of the glucose-regulated protein 78 (GRP78) gene. The N-terminal region of the human GRP78 gene was fused to the NanoLuc gene and aligned with the puromycin-resistant gene through the 2A peptide sequence and used as a knock-in vector. The selected cells responded to both pharmacological and genetic ER stress and show NanoLuc-based CRISPR/Cas9 system is a very useful tool to isolate gene-edited cells and to characterize the endogenous promoter activity for genes of interest.

A Comparative Analysis of the Molecular Features of MANF and CDNF.

Cerebral dopamine neurotrophic factor (CDNF) is a paralogous protein of mesencephalic astrocyte-derived neurotrophic factor (MANF). Both proteins have been reported to show a common cytoprotective effect on dopaminergic neurons as a secretory protein containing the KDEL-like motif of the ER retrieval signal at the C-terminus, RTDL in MANF and [Q/K]TEL in CDNF among many species, although functions of paralogous proteins tend to differ from each other. In this study, we focused on post-translational regulations of their retention in the endoplasmic reticulum (ER) and secretion and performed comparative experiments on characterization of mouse MANF and mouse CDNF according to our previous report about biosynthesis and secretion of mouse MANF using a NanoLuc system.

A highly sensitive assay of IRE1 activity using the small luciferase NanoLuc: Evaluation of ALS-related genetic and pathological factors.

Activation of inositol-requiring enzyme 1 (IRE1) due to abnormal conditions of the endoplasmic reticulum (ER) is responsible for the cleavage of an unspliced form of X-box binding protein 1 (uXBP1), producing its spliced form (sXBP1). To estimate IRE1 activation, several analytical procedures using green fluorescence protein and firefly luciferase have been developed and applied to clarify the roles of IRE1-XBP1 signaling pathways during development and disease progression. In this study, we established a highly sensitive assay of IRE1 activity using a small luciferase, NanoLuc, which has approximately 100-fold higher activity than firefly luciferase.

Characterization of the Role of MANF in Regulating the Secretion of CRELD2.

We recently demonstrated that the secretion of two novel endoplasmic reticulum (ER) stress-inducible proteins, cysteine-rich with epidermal growth factor (EGF)-like domains 2 (CRELD2) and mesencephalic astrocyte-derived neurotrophic factor (MANF), are oppositely regulated by the overexpression of 78 kDa glucose-regulated protein (GRP78). In the present study, we found that the co-transfection of CRELD2 and MANF remarkably enhanced the secretion of CRELD2 without affecting the expression level of GRP78. To identify the structural features of CRELD2 and MANF involved in this process, we generated several CRELD2 and MANF expression constructs.

A sensitive assay for the biosynthesis and secretion of MANF using NanoLuc activity.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) has been reported to prevent neuronal cell death caused by certain stimuli. Accordingly, the molecular features of MANF have been intensively investigated since the reporting of its cytoprotective actions. In addition to the characterization of the transcriptional regulation of MANF under pathophysiological conditions, it is important to understand its intracellular transport and secretion after translation.