Vitamin D-dependent protein of smooth endoplasmic reticulum membranes of rat enterocytes

DEAE-cellulose chromatography and SDS-PAAG electrophoresis were used to study proteins from endoplasmic reticulum of smooth and rough enterocyte membranes in the vitamin D-replete and deficient rat. The membrane-bound vitamin-D-dependent protein with a molecular weight of 21-25 kDa is shown to consist of the endoplasmic reticulum of smooth membranes. The intensive binding of calcium by these membranes is established.     

Apoptosis induced by endoplasmic reticulum stress involved in diabetic kidney disease

Endoplasmic reticulum stress has been suggested to play a crucial role in the pathogenesis of diabetic complications. However, whether it is involved in the renal injury of diabetic nephropathy is still not known. We investigated the involvement of ER-associated apoptosis in kidney disease of streptozocin (STZ)-induced diabetic rats. We used albuminuria examination, hematoxylin & eosin (H&E) staining and TUNEL analysis to identify the existence of diabetic nephropathy and enhanced apoptosis. We performed immunohistochemistry, Western blot, and real-time PCR to analyze indicators of ER molecule chaperone and ER-associated apoptosis. GRP78, the ER chaperone, was up-regulated significantly in diabetic kidney compared to control. Furthermore, three hallmarks of ER-associated apoptosis, C/EBP homologous protein (CHOP), c-JUN NH2-terminal kinase (JNK) and caspase-12, were found to have activated in the diabetic kidney. Taken together, those results suggested that apoptosis induced by ER stress occurred in diabetic kidney, which may contribute to the development of diabetic nephropathy.     

Characterization of mouse endoplasmic reticulum methionine-R-sulfoxide reductase

Methionine-R-sulfoxide reductases (MsrBs) catalyze a stereospecific reduction of methionine-R-sulfoxides to methionines in proteins. Mammals possess three MsrB genes. MsrB1 (SelR) is a selenoprotein located in the cytosol and nucleus, MsrB2 (CBS-1) is a mitochondrial protein, and MsrB3 is a recently identified protein with an unusual localization pattern. Human MsrB3 occurs in two protein forms, MsrB3A and MsrB3B, which can be targeted to the endoplasmic reticulum (ER) and mitochondria, respectively. These forms are generated by alternative first exon splicing that introduces contrasting N-terminal signal peptides. Herein, we characterized mouse MsrB3 and found no evidence of alternative splicing of its gene. The ER signal was located upstream of the predicted mitochondrial signal sequence in a single coding region, whose product was targeted to the ER. Although the mitochondrial signal could function if placed at the N-terminus, it did not target MsrB3 to mitochondria as part of the entire coding region. In addition, immunoblot assays detected no mitochondrial MsrB3 in examined mouse tissues. The data suggest that, in mice, MsrB3 is largely or exclusively an ER-resident protein, and that the reduction of methionine-R-sulfoxides in different cellular compartments is provided by individual MsrB isozymes.     

Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress

The ability to respond to perturbations in endoplasmic reticulum (ER) function is a fundamentally important property of all cells, but ER stress can also lead to apoptosis. In settings of chronic ER stress, the associated apoptosis may contribute to pathophysiological processes involved in a number of prevalent diseases, including neurodegenerative diseases, diabetes, atherosclerosis and renal disease. The molecular mechanisms linking ER stress to apoptosis are the topic of this review, with emphases on relevance to pathophysiology and integration and complementation among the various apoptotic pathways induced by ER stress.     

Changes in endoplasmic reticulum during spermiogenesis in the mouse

Summary Changes in the endoplasmic reticulum of mouse spermatids during spermiogenesis were examined by scanning electron microscopy, applying the OsO₄-DMSO-OsO₄ method, which permits 3-dimensional observation of cell organelles. At the same time, the endoplasmic reticulum was stained selectively by the Ur-Pb-Cu method, and 0.5 m-thick sections were prepared for observation by transmission electron microscopy. The results demonstrated stereoscopically the mode of disappearance of the endoplasmic reticulum. In spermatids of the early maturation phase, the endoplasmic reticulum was of uniform diameter, branched and anastomosed, forming a complicated three-dimensional network throughout the cytoplasm. A two-dimensional net was also noted to have formed just beneath the plasma membrane and about Sertoli cell processes invaginating the spermatid cytoplasm. As spermiogenesis progressed, the spread-out endoplasmic reticulum gradually aggregated to form a condensed, glomerulus-like structure consisting of a very thin endoplasmic reticulum connected to the surrounding endoplasmic reticulum. This structure corresponds to the so-called radial body. Thus, the endoplasmic reticulum may aggregate, condense, be transformed into a radial body, and be removed from the cytoplasm. The two-dimensional endoplasmic reticulum-net, just beneath the plasma membrane and surrounding processes of Sertoli cells, disappeared in spaces where the three-dimensional endoplasmic reticulum network was scarce. Both the two-dimensional endoplasmic reticulum-net structure and the three-dimensional endoplasmic reticulum network disappeared at the same time, indicating that they may be closely related.     

HDLs inhibit endoplasmic reticulum stress and autophagic response induced by oxidized LDLs

The apoptotic effect of oxidized LDLs (oxLDLs) is mediated through a complex sequence of signaling events involving a deregulation of the cytosolic Ca(2+) homeostasis. OxLDLs also trigger ER stress that may lead to cellular dysfunction and apoptosis, through the activation of the IRE1α/c-Jun N-terminal kinase pathway. Moreover, ER stress and oxidized lipids have been shown to trigger autophagy. The antiatherogenic high-density lipoproteins (HDLs) display protective effects against oxLDLs toxicity. To more deeply investigate the mechanisms mediating the protective effects of HDLs, we examined whether ER stress and autophagy were implicated in oxLDLs-induced apoptosis and whether HDLs prevented these stress processes. We report that, in human endothelial cells, HDLs prevent the oxLDL-induced activation of the ER stress sensors IRE1α, eIF2α and ATF6 and subsequent activation of the proapoptotic mediators JNK and CHOP. OxLDLs also trigger the activation of autophagy, as assessed by LC3 processing and Beclin-1 expression. The autophagic process is independent of the proapoptotic arms of ER stress, but Beclin-1 contributes to PS exposure and subsequent phagocytosis of oxLDLs exposed cells. Induction of autophagy and PS exposure by oxLDLs is prevented by HDLs. Finally, the cytosolic Ca(2+) deregulation triggered by oxLDLs is a common signaling pathway that mediates ER stress-induced cell death and autophagy, all these events being blocked by HDLs.     

Inhibition of endoplasmic reticulum stress improves mouse embryo development

X-box binding protein-1 (XBP-1) is an important regulator of a subset of genes during endoplasmic reticulum (ER) stress. In the current study, we analyzed endogenous XBP-1 expression and localization, with a view to determining the effects of ER stress on the developmental competency of preimplantation embryos in mice. Fluorescence staining revealed that functional XBP-1 is localized on mature oocyte spindles and abundant in the nucleus at the germinal vesicle (GV) stage. However, in preimplantation embryos, XBP-1 was solely detected in the cytoplasm at the one-cell stage. The density of XBP-1 was higher in the nucleus than the cytoplasm at the two-cell, four-cell, eight-cell, morula, and blastocyst stages. Furthermore, RT-PCR analysis confirmed active XBP-1 mRNA splicing at all preimplantation embryo stages, except the one-cell stage. Tunicamycin (TM), an ER stress inducer used as a positive control, promoted an increase in the density of nuclear XBP-1 at the one-cell and two-cell stages. Similarly, culture medium supplemented with 25 mM sorbitol displayed a remarkable increase active XBP-1 expression in the nuclei of 1-cell and 2-cell embryos. Conversely, high concentrations of TM or sorbitol led to reduced nuclear XBP-1 density and significant ER stress-induced apoptosis. Tauroursodeoxycholic acid (TUDCA), a known inhibitor of ER stress, improved the rate of two-cell embryo development to blastocysts by attenuating the expression of active XBP-1 protein in the nucleus at the two-cell stage. Our data collectively suggest that endogenous XBP-1 plays a role in normal preimplantation embryonic development. Moreover, XBP-1 splicing is activated to generate a functional form in mouse preimplantation embryos during culture stress. TUDCA inhibits hyperosmolar-induced ER stress as well as ER stress-induced apoptosis during mouse preimplantation embryo development.     

A transgenic mouse model for monitoring endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress is caused by the accumulation of unfolded proteins in the ER lumen, and is associated with vascular and neurodegenerative diseases. Although the connection between ER stress and some disease-related proteins has been studied using animal models of these diseases, no in vivo data concerning ER stress are available. Here we report a new method for monitoring ER stress in vivo, based on XBP-1 mRNA splicing by inositol requiring-1 (IRE-1) during ER stress. The stress indicator was constructed by fusing XBP-1 and venus, a variant of green fluorescent protein. During stress, the spliced indicator mRNA is translated into an XBP-1-venus fusion protein, which can be detected by its fluorescence. We used transgenic animals expressing the ER stress indicator to show that it can be used to monitor physiological and pathological ER stress in vivo.     

Calnexin is involved in apoptosis induced by endoplasmic reticulum stress in the fission yeast

Stress conditions affecting the functions of the endoplasmic reticulum (ER) cause the accumulation of unfolded proteins. ER stress is counteracted by the unfolded-protein response (UPR). However, under prolonged stress the UPR initiates a proapoptotic response. Mounting evidence indicate that the ER chaperone calnexin is involved in apoptosis caused by ER stress. Here, we report that overexpression of calnexin in Schizosaccharomyces pombe induces cell death with apoptosis markers. Cell death was partially dependent on the Ire1p ER-stress transducer. Apoptotic death caused by calnexin overexpression required its transmembrane domain (TM), and involved sequences on either side of the ER membrane. Apoptotic death caused by tunicamycin was dramatically reduced in a strain expressing endogenous levels of calnexin lacking its TM and cytosolic tail. This demonstrates the involvement of calnexin in apoptosis triggered by ER stress. A genetic screen identified the S. pombe homologue of the human antiapoptotic protein HMGB1 as a suppressor of apoptotic death due to calnexin overexpression. Remarkably, overexpression of human calnexin in S. pombe also provoked apoptotic death. Our results argue for the conservation of the role of calnexin in apoptosis triggered by ER stress, and validate S. pombe as a model to elucidate the mechanisms of calnexin-mediated cell death.     

Laminated cisternae of the rough endoplasmic reticulum induced by coronavirus MHV-A59 infection

The infection of murine fibroblasts of the sac- line with a coronavirus, mouse hepatitis virus strain A59 (MHV-A59), results in a novel modification to some cisternae of the rough endoplasmic reticulum (RER). From 8 hours post infection (h.p.i.) we see in thin sections pairs of cisternae closely, stably and uniformly aligned. Serial sectioning shows that the regions of pairing or lamination extend for many thousands of nm in two dimensions, with the spacing between the juxtaposed membranes remaining very uniform at about 18 nm. These structures appear coincident with the onset of accumulation of the viral glycoprotein E1 in the RER membrane but 2 hours after the viral glycoprotein E2 can first be detected there. Ribosomes are excluded from the paired cisternal surfaces, while budding of progeny virions has never been seen at the cisternal membranes facing the cytosol, although ribosomes bind there. The lumina of paired cixternae are usually devoid of virions which, however, accumulate in areas where the paired cisternae diverge. Electron immunocytochemistry shows that both E1 and E2 glycoproteins are abundant in the paired cisternae. Following labelling for the E1 glycoprotein we see a periodic fine structure, rows of "beads" with a centre to centre spacing of about 7.5 nm, in the region between the paired membranes. In oblique sections of this region in cells fixed as if for the immunoperoxidase labelling, but omitting all its steps we see parallel rows of "beads" separated by about 7 nm. We suggest that the membrane spanning viral glycoprotein E1 together with viral nucleocapsids may be involved in laminating cisternae of the RER.     

Lipids and proteolipids in membranes of the smooth endoplasmic reticulum of rat enterocytes. Vitamin D-dependent calcium binding

The lipid and proteolipid composition of endoplasmic reticulum smooth membranes from rat enterocytes in normalcy and under D-hypovitaminosis is studied to show the Ca2+-binding with these components. These membranes in vitamin D-deficiency are established to differ in the cholesterol content. Proteolipids 1 and 2 are isolated from the lipid fraction of membranes. Their amino acid composition is determined. The vitamin D-dependent binding of Ca by the proteolipid fraction is shown.     

ERp16, an endoplasmic reticulum-resident thiol-disulfide oxidoreductase: biochemical properties and role in apoptosis induced by endoplasmic reticulum stress

We have characterized the properties and putative role of a mammalian thioredoxin-like protein, ERp16 (previously designated ERp18, ERp19, or hTLP19). The predicted amino acid sequence of the 172-residue human protein contains an NH(2)-terminal signal peptide, a thioredoxin-like domain with an active site motif (CGAC), and a COOH-terminal endoplasmic reticulum (ER) retention sequence (EDEL). Analyses indicated that the mature protein (comprising 146 residues) is generated by cleavage of the 26-residue signal peptide and is localized in the lumen of the ER. Biochemical experiments with the recombinant mature protein revealed it to be a thioldisulfide oxidoreductase. Its redox potential was about -165 mV; its active site cysteine residue Cys(66) was nucleophilic with a pK(a) value of approximately 6.6; it catalyzed the formation, reduction, and isomerization of disulfide bonds, with the unusual CGAC active site motif being responsible for these activities; and it existed as a dimer and underwent a redox-dependent conformational change. The observations that the redox potential of ERp16 (-165 mV) was within the range of that of the ER (-135 to -185 mV) and that ERp16 catalyzed disulfide isomerization of scrambled ribonuclease A suggest a role for ERp16 in protein disulfide isomerization in the ER. Expression of ERp16 in HeLa cells inhibited the induction of apoptosis by agents that elicit ER stress, including brefeldin A, tunicamycin, and dithiothreitol. In contrast, expression of a catalytically inactive mutant of ERp16 potentiated such apoptosis, as did depletion of ERp16 by RNA interference. Our results suggest that ERp16 mediates disulfide bond formation in the ER and plays an important role in cellular defense against prolonged ER stress.