No abstract

Aberrant protein folding beyond the capacity of endoplasmic reticulum (ER) quality control leads to stress response in the ER. The Lys-Asp-Glu-Leu (KDEL) receptor, a retrieval receptor for ER chaperones in the early secretory pathway, contributes to ER quality control. To elucidate the function of the KDEL receptor in vivo, we established transgenic mice expressing a mutant KDEL receptor. We found that the mutant KDEL receptor sensitized cells to ER stress and that the mutant mice developed dilated cardiomyopathy. Ultrastructural analyses revealed expanded sarcoplasmic reticulums and protein aggregates that obstructed the adjacent transverse tubules of the mutant cardiomyocytes. Cardiomyocytes from the mutant mice were sensitive to ER stress when treated with tunicamycin and showed a functional defect in the L-type Ca 2؉ current. We observed ubiquitinated protein aggregates, enhanced expression of CHOP (a death-related transcriptional factor expressed upon ER stress), and apoptosis in the mutant hearts. These findings suggest that impairment of the KDEL receptor disturbs ER quality control, resulting in accumulation of misfolded proteins in the ER in an in vivo system, and that the dilated cardiomyopathy found in the mutant KDEL receptor transgenic mice is associated with ER stress.The endoplasmic reticulum (ER) provides a folding environment for newly synthesized secretory and membrane proteins (10). Aberrant protein folding due to extracellular stimuli, such as ischemia and oxidative stress, and genetic mutation lead to the accumulation of misfolded proteins in the ER, which in turn evokes the unfolded protein response (43), which reduces the amount of misfolded proteins by inducing the production of ER chaperones that promote protein folding, reducing general protein synthesis (16) and enhancing the degradation of misfolded proteins via a ubiquitin-proteasome system termed ER-associated degradation (7, 9, 60). The persistent accumulation of misfolded proteins beyond the capacity of ER quality control causes cellular dysfunction and cell death (24,25,46). This process is involved in diverse human disorders, including diabetes mellitus (14, 42) and neurodegenerative diseases such as Alzheimer's (23) and Parkinson's (20).Misfolded proteins had been believed to remain in the ER, but recent genetic analyses in Saccharomyces cerevisiae have indicated that the unfolded protein response involves the whole secretory pathway (56) and that some misfolded proteins require transport between the ER and the Golgi complex for ER-associated degradation (17,41,53,58). In addition, certain misfolded proteins in mammalian cells have also been reported to exit the ER and recycle between the ER and post-ER compartments, associating with ER chaperones. The KDEL receptor mediates this retrieval, suggesting that the secretion of misfolded proteins from the ER and their retrieval may contribute to ER quality control (12, 62).The KDEL receptor has been identified as a retrieval receptor for luminal ER chaperones that have a carboxyl-te...

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR), which alleviates protein overload in the secretory pathway. Although the UPR is activated under diverse pathological conditions, its physiological role during development and in adulthood has not been fully elucidated. Binding immunoglobulin protein (BiP) is an ER chaperone, which is central to ER function. We produced knock-in mice expressing a mutant BiP lacking the retrieval sequence to cause a defect in ER function without completely eliminating BiP. In embryonic fibroblasts, the UPR compensated for mutation of BiP. However, neonates expressing mutant BiP suffered respiratory failure due to impaired secretion of pulmonary surfactant by alveolar type II epithelial cells. Expression of surfactant protein (SP)-C was reduced and the lamellar body was malformed, indicating that BiP plays a critical role in the biosynthesis of pulmonary surfactant. Because pulmonary surfactant requires extensive post-translational processing in the secretory pathway, these findings suggest that in secretory cells, such as alveolar type II cells, the UPR is essential for managing the normal physiological ER protein overload that occurs during development. Moreover, failure of this adaptive mechanism may increase pulmonary susceptibility to environmental insults, such as hypoxia and ischemia, ultimately leading to neonatal respiratory failure.

The carboxyl-terminal cholinesterase-like (ChEL) domain of thyroglobulin (Tg) has been identified as critically important in Tg export from the endoplasmic reticulum. In a number of human kindreds suffering from congenital hypothyroidism, and in the cog congenital goiter mouse and rdw rat dwarf models, thyroid hormone synthesis is inhibited because of mutations in the ChEL domain that block protein export from the endoplasmic reticulum. We hypothesize that Tg forms homodimers through noncovalent interactions involving two predicted ␣-helices in each ChEL domain that are homologous to the dimerization helices of acetylcholinesterase. This has been explored through selective epitope tagging of dimerization partners and by inserting an extra, unpaired Cys residue to create an opportunity for intermolecular disulfide pairing. We show that the ChEL domain is necessary and sufficient for Tg dimerization; specifically, the isolated ChEL domain can dimerize with full-length Tg or with itself. Insertion of an N-linked glycan into the putative upstream dimerization helix inhibits homodimerization of the isolated ChEL domain. However, interestingly, co-expression of upstream Tg domains, either in cis or in trans, overrides the dimerization defect of such a mutant. Thus, although the ChEL domain provides a nidus for Tg dimerization, interactions of upstream Tg regions with the ChEL domain actively stabilizes the Tg dimer complex for intracellular transport.

There is a rapidly growing demand for female animals in preclinical animal, and thus it is necessary to determine animals' estrous cycle stages from vaginal smear cytology. However, the determination of estrous stages requires extensive training, takes a long time, and is costly; moreover, the results obtained by human examiners may not be consistent. Here, we report a machine learning model trained with 2,096 microscopic images that we named the "Stage Estimator of estrous Cycle of RodEnt using an Image-recognition Technique (SECREIT)." With the test dataset (736 images), SECREIT achieved area under the receiver-operating-characteristic curve of 0.962 or more for each estrous stage. A test using 100 images showed that SECREIT provided correct classification that was similar to that provided by two human examiners (SECREIT: 91%, Human 1: 91%, Human 2: 79%) in 11 s. The SECREIT can be a first step toward accelerating the research using female rodents. Knowledge of the precise stages of the estrous cycle is very important for interpretations of female animals' data. Compared to men, women have a high lifetime incidence of several mental illnesses, including depression, posttraumatic stress disorders, generalized anxiety, and eating disorders 1 , but preclinical animal investigations for these illnesses have use mainly males in part because of the volatility of female animals' experimental data that cannot be separated from their estrous cycle. Several research groups have stated that it is difficult to apply the findings from males' preclinical results to women's medicine 2. Indeed, the estrous cycle affects the expression of genes 3 , proteins 4,5 , electrophysiological properties 6,7 , behaviors 8,9 , and drug effects 10. In 2015, the U.S. National Institutes of Health (NIH) announced that when conducting NIH-funded research, researchers should study both sexes 11. The number of preclinical studies using female animals is thus gradually increasing. The estrous cycle in rodents is generally divided into three or four stages 12 , and the cycle is 4-5 days: Diestrus (D) → proestrus (P) → estrus (E) → (metestrus) →. In many studies of rodents, the estrous cycle stage of each animal has been determined by vaginal cytology. Each stage is decided based on the type, number, shape, size, and proportion of cells in a vaginal smear 12-17 (Fig. 1a-c). Briefly, stage D was identified by the presence of leukocytes and nucleated cells with or without a few cornified cells. Stage P was identified by the presence of nucleated epithelial cells and cornified cells without leukocytes. Stage E was identified by the presence of nucleated epithelial cells without leukocytes or cornified cells. Vaginal cytology is also used to decide whether an ovariectomy in a rodent is successful 18,19 (Fig. 1d). However, determining the estrous stage of a rodent by using vaginal cytology evaluated by a human examiner has some problems: (1) a long training period is required in order to become skillful; (2) it takes a long time to determine the estrou...