Genome-wide identification and gene expression profiling of ubiquitin ligases for endoplasmic reticulum protein degradation

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a mechanism by which unfolded proteins that accumulate in the ER are transported to the cytosol for ubiquitin–proteasome-mediated degradation. Ubiquitin ligases (E3s) are a group of enzymes responsible for substrate selectivity and ubiquitin chain formation. The purpose of this study was to identify novel E3s involved in ERAD. Thirty-seven candidate genes were selected by searches for proteins with RING-finger motifs and transmembrane regions, which are the major features of ERAD E3s. We performed gene expression profiling for the identified E3s in human and mouse tissues. Several genes were specifically or selectively expressed in both tissues; the expression of four genes (RNFT1, RNF185, CGRRF1 and RNF19B) was significantly upregulated by ER stress. To determine the involvement of the ER stress-responsive genes in ERAD, we investigated their ER localisation, in vitro autoubiquitination activity and ER stress resistance. All were partially localised to the ER, whereas CGRRF1 did not possess E3 activity. RNFT1 and RNF185, but not CGRRF1 and RNF19B, exhibited significant resistance to ER stressor in an E3 activity-dependent manner. Thus, these genes are possible candidates for ERAD E3s.This study was supported by Grants-in-Aid for Scientific Research (KAKENHI) 15K21706, 26460099, 24300135, 22020032, 25251014, 15K15067, 15K20001, 15K18377 and 15K19516 from the Ministry of Education, Culture, Sports, Science and Technology, Japan and also supported by the Takeda Science Foundation. We thank H. Hishigaki and Otsuka GEN Research Institute for bioinformatic analysis. We also thank M. Minami and T. Uehara for the helpful discussions. We are grateful to T. Yoshikawa, T. Ike, Y. Maeoka, Y. Wada and Z. Cao for their technical assistance. The authors would like to thank Enago (www.enago.jp) for the English language review

Inhibition of Casein Kinase 2 Modulates XBP1-GRP78 Arm of Unfolded Protein Responses in Cultured Glial Cells

Stress signals cause abnormal proteins to accumulate in the endoplasmic reticulum (ER). Such stress is known as ER stress, which has been suggested to be involved in neurodegenerative diseases, diabetes, obesity and cancer. ER stress activates the unfolded protein response (UPR) to reduce levels of abnormal proteins by inducing the production of chaperon proteins such as GRP78, and to attenuate translation through the phosphorylation of eIF2α. However, excessive stress leads to apoptosis by generating transcription factors such as CHOP. Casein kinase 2 (CK2) is a serine/threonine kinase involved in regulating neoplasia, cell survival and viral infections. In the present study, we investigated a possible linkage between CK2 and ER stress using mouse primary cultured glial cells. 4,5,6,7-tetrabromobenzotriazole (TBB), a CK2-specific inhibitor, attenuated ER stress-induced XBP-1 splicing and subsequent induction of GRP78 expression, but was ineffective against ER stress-induced eIF2α phosphorylation and CHOP expression. Similar results were obtained when endogenous CK2 expression was knocked-down by siRNA. Immunohistochemical analysis suggested that CK2 was present at the ER. These results indicate CK2 to be linked with UPR and to resist ER stress by activating the XBP-1-GRP78 arm of UPR

Possible Involvement of Ubiquitin Ligase HRD1 Insolubilization in Amyloid β Generation

Endoplasmic reticulum (ER)-associated degradation (ERAD) selectively retro-transports and degrades unfolded proteins accumulated in the ER. We have demonstrated that the ubiquitin ligase HRD1 involved in ERAD was significantly decreased in the cerebral cortex of Alzheimer’s disease patients. Furthermore, the HRD1 level was negatively correlated with amyloid β (Aβ) production levels. Here we found that the HRD1 protein level decrease was due to its insolubilization. Moreover, these protein levels extracted from detergent insoluble fraction were positively correlated with those of SEL1L and Aβs (Aβ40 and Aβ42). Thus, the insolubilization-induced decrease in the HRD1 and SEL1L levels might involve in Aβ generation.This study was supported by Grants-in-Aid for Science Research (KAKENHI) 21790089, 21300142, and 20659013 from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by the Research Foundation for Pharmaceutical Sciences

A different pathway in the endoplasmic reticulum stress-induced expression of human HRD1 and SEL1 genes

Human HRD1 and SEL1 are components of endoplasmic reticulum-associated degradation (ERAD), which is a retrograde transport mechanism from the ER to the cytosol for removing unfolded proteins. The expression of HRD1 and SEL1 was induced by ER stress-inducing agents and overexpression of both ER stress-responsive transcription factors, ATF6 and XBP1. Inhibition of IRE1 and ATF6 revealed that ER stress-induced HRD1 and SEL1 expressions are mediated by IRE1-XBP1- and ATF6-dependent pathways, respectively. These results suggest that the ER stress-induced ERAD gene expressions are mediated by different pathways, which are attributed to the differences in the promoter regions

Inhibitory effect of 4-(2-aminoethyl)-benzenesulfonyl fluoride, a serine protease inhibitor, on PI3K inhibitor-induced CHOP expression

Endoplasmic reticulum stress contributes to several diseases such as neurodegenerative disorders and diabetes. In the previous report, we found that phosphatidylinositol 3-kinase (PI3K) down-regulation is important for inducing CHOP expression, an endoplasmic reticulum stress-induced transcription factor. In the present study, we investigated the effect of 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor, on PI3K inhibitor-induced CHOP expression. We found that AEBSF completely inhibited PI3K inhibitor-induced CHOP expression at both mRNA and protein levels. It is suggested that AEBSF is an important drug from a pharmacological point of view and the results may have important implications for understanding endoplasmic reticulum stress-related diseases

Endoplasmic Reticulum Stress and Parkinson’s Disease: The Role of HRD1 in Averting Apoptosis in Neurodegenerative Disease

Endoplasmic reticulum (ER) stress has been known to be involved in the pathogenesis of various diseases, particularly neurodegenerative disorders such as Parkinson’s disease (PD). We previously identified the human ubiquitin ligase HRD1 that is associated with protection against ER stress and its associated apoptosis. HRD1 promotes the ubiquitination and degradation of Parkin-associated endothelin receptor-like receptor (Pael-R), an ER stress inducer and causative factor of familial PD, thereby preventing Pael-R-induced neuronal cell death. Moreover, upregulation of HRD1 by the antiepileptic drug zonisamide suppresses 6-hydroxydopamine-induced neuronal cell death. We review recent progress in the studies on the mechanism of ER stress-induced neuronal death related to PD, particularly focusing on the involvement of HRD1 in the prevention of neuronal death as well as a potential therapeutic approach for PD based on the upregulation of HRD1