Akio Tsuru

Diphtheria toxin receptor–mediated conditional and targeted cell ablation in transgenic mice

Specific cell ablation is a useful method for analyzing the in vivo function of cells. We have developed a simple and sensitive method for conditional cell ablation in transgenic mice, called “toxin receptor–mediated cell knockout.” We expressed the diphtheria toxin (DT) receptor in transgenic mice using a hepatocyte-specific promoter and found that injection of DT caused fulminant hepatitis. Three independently established transgenic lines demonstrated a good correlation between the sensitivity of hepatocytes to DT and the expression level of the DT receptors. Moreover, the degree of hepatocyte damage was easily controlled over a wide range of doses of injected DT without any obvious abnormalities in other cells or tissues. This system is useful for generating mouse models of disease and for studying the recovery or regeneration of tissues from cell damage or loss. As DT is a potent inhibitor of protein synthesis in both growing and non-growing cells, the method is applicable to a wide range of cells and tissues in mice or in other DT-insensitive animals.

Translational control by the ER transmembrane kinase/ribonuclease IRE1 under ER stress

Under conditions of endoplasmic reticulum (ER) stress, mammalian cells induce both translational repression and the unfolded protein response that transcriptionally activates genes encoding ER-resident molecular chaperones. To date, the only known pathway for translational repression in response to ER stress has been the phosphorylation of eIF-2α by the double-stranded RNA-activated protein kinase (PKR) or the transmembrane PKR-like ER kinase (PERK). Here we report another pathway in which the ER transmembrane kinase/ribonuclease IRE1β induces translational repression through 28S ribosomal RNA cleavage in response to ER stress. The evidence suggests that both pathways are important for efficient translational repression during the ER stress response.

Negative feedback by IRE1β optimizes mucin production in goblet cells

In mammals, the prototypical endoplasmic reticulum (ER) stress sensor inositol-requiring enzyme 1 (IRE1) has diverged into two paralogs. IRE1α is broadly expressed and mediates the unconventional splicing of X-box binding protein 1 (XBP1) mRNA during ER stress. By contrast, IRE1β is expressed selectively in the digestive tract, and its function remains unclear. Here, we report that IRE1β plays a distinctive role in mucin-secreting goblet cells. In IRE1β−/− mice, aberrant mucin 2 (MUC2) accumulated in the ER of goblet cells, accompanied by ER distension and elevated ER stress signaling such as increased XBP1 mRNA splicing. In contrast, conditional IRE1α−/− mice showed no such ER distension but a marked decrease in spliced XBP1 mRNA. mRNA stability assay revealed that MUC2 mRNA was greatly stabilized in IRE1β−/− mice. These findings suggest that in goblet cells, IRE1β, but not IRE1α, promotes efficient protein folding and secretion in the ER by optimizing the level of mRNA encoding their major secretory product, MUC2.

RNase domains determine the functional difference between IRE1α and IRE1β

Endoplasmic reticulum (ER) stress is associated with the functional disorder of the ER. During conditions of ER stress, cells induce at least two responses to maintain ER function: transcriptional upregulation of ER quality control genes, and translational attenuation of protein synthesis. Induction of ER quality control proteins is mediated by IRE1α, which activates the transcription factor XBP1 via an unconventional splicing event, while a partial translational attenuation is mediated by IRE1β. Here, we show by both in vivo and in vitro analyses that the RNase domain of IRE1 determines the functional specificities of each of these isoforms.

Mammalian ER stress sensor IRE1β specifically down‐regulates the synthesis of secretory pathway proteins

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress. The ER stress sensor inositol requiring enzyme‐1beta (IRE1β), which is specifically expressed in intestinal epithelial cells, is thought to be involved in translational repression. However, its mechanism of action is not fully understood. Using a reporter that can evaluate and distinguish between translation efficiency in the cytosol and on the ER membrane, we show here that IRE1β represses translation on the ER membrane but not in the cytosol, and that this selective repression depends on the RNase activity of IRE1β.

Novel mechanism of enhancing IRE1α-XBP1 signalling via the PERK-ATF4 pathway

Mammalian inositol-requiring enzyme 1α (IRE1α) is the most conserved of all endoplasmic reticulum (ER) stress sensors, which includes activating transcription factor (ATF) 6 and double-stranded RNA-dependent protein kinase (PKR)-like ER kinase (PERK). IRE1α has been known to splice X-box binding protein 1 (XBP1) mRNA, which is induced by ATF6 under ER stress. This spliced XBP1 mRNA is translated into the active transcription factor that promotes the expression of specific genes to alleviate ER stress. Herein, we report that in addition to the induction of XBP1 expression by ATF6, IRE1α expression is induced by ATF4, which is downstream of PERK, under ER stress. Increased IRE1α expression results in a higher splicing ratio of XBP1 mRNA. This effect was not transient and affected not only the intensity but also the duration of the activated state of this pathway. These multiple regulatory mechanisms may modulate the response to various levels or types of ER stress.

Identification of a novel mammalian endoplasmic reticulum-resident KDEL protein using an EST database motif search.

Several endoplasmic reticulum (ER)-resident proteins contain a unique C-terminal sequence (KDEL) which is required for the retention of these proteins in the ER. By searching a mouse EST database for records containing the nucleotide sequence encoding the KDEL motif, we extracted cDNAs encoding putative novel ER-resident proteins in addition to all of the known ER proteins bearing the KDEL motif. Using the sequence information obtained by this database search, we cloned the cDNA encoding a novel KDEL motif-bearing protein, ER protein 58 (EP58), sharing no significant homology to any of the known ER-resident proteins. Subcellular localization of EP58 in the ER was confirmed by cytoimmunofluorescence studies using epitope-tagged EP58. The EP58 gene was primarily expressed in embryo, placenta, and adult heart. Neither heat shock nor ER stress as tested here was sufficient to induce expression of the EP58 gene. A putative role of the N-terminal half of EP58 in protein-protein interaction is suggested by its similarity to the filamin rod domain. Similarity of the EP58 sequence with bacterial and fungus proteins suggests a possible role for EP58 in polysaccharide biosynthesis.

Identification of the redox partners of ERdj5/JPDI, a PDI family member, from an animal tissue

ERdj5 (also known as JPDI) is a member of PDI family conserved in higher eukaryotes. This protein possesses an N-terminal J domain and C-terminal four thioredoxin domains each having a redox active site motif. Despite the insights obtained at the cellular level on ERdj5, the role of this protein in vivo is still unclear. Here, we present a simple method to purify and identify the disulfide-linked complexes of this protein efficiently from a mouse tissue. By combining acid quenching and thiol-alkylation, we identified a number of potential redox partners of ERdj5 from the mouse epididymis. Further, we show that ERdj5 indeed interacted with two of the identified proteins via formation of intermolecular disulfide bond. Thus, this approach enabled us to detect and identify redox partners of a PDI family member from an animal tissue.