EpCAM associates with endoplasmic reticulum aminopeptidase 2 (ERAP2) in breast cancer cells

Epithelial cell adhesion molecule (EpCAM) is an epithelial and cancer cell “marker” and there is a cumulative and growing evidence of its signaling role. Its importance has been recognized as part of the breast cancer stem cell phenotype, the tumorigenic breast cancer stem cell is EpCAM⁺. In spite of its complex functions in normal cell development and cancer, relatively little is known about EpCAM-interacting proteins. We used breast cancer cell lines and performed EpCAM co-immunoprecipitation followed by mass spectrometry in search for novel potentially interacting proteins. The endoplasmic reticulum aminopeptidase 2 (ERAP2) was found to co-precipitate with EpCAM and to co-localize in the cytoplasm/ER and the plasma membrane. ERAP2 is a proteolytic enzyme set in the endoplasmic reticulum (ER) where it plays a central role in the trimming of peptides for presentation by MHC class I molecules. Expression of EpCAM and ERAP2 in vitro in the presence of dog pancreas rough microsomes (ER vesicles) confirmed N-linked glycosylation, processing in ER and the size of EpCAM. The association between ERAP2 and EpCAM is a unique and novel finding that provides new ideas on EpCAM processing and on how antigen presentation may be regulated in cancer.     

Altered localization of amyloid precursor protein under endoplasmic reticulum stress

Recent reports have shown that the endoplasmic reticulum (ER) stress is relevant to the pathogenesis of Alzheimer disease. Following the amyloid cascade hypothesis, we therefore attempted to investigate the effects of ER stress on amyloid-beta peptide (Abeta) generation. In this study, we found that ER stress altered the localization of amyloid precursor protein (APP) from late compartments to early compartments of the secretory pathway, and decreased the level of Abeta 40 and Abeta 42 release by beta- and gamma-cutting. Transient transfection with BiP/GRP78 also caused a shift of APP and a reduction in Abeta secretion. It was revealed that the ER stress response facilitated binding of BiP/GRP78 to APP, thereby causing it to be retained in the early compartments apart from a location suitable for the cleavages of Abeta. These findings suggest that induction of BiP/GRP78 during ER stress may be one of the regulatory mechanisms of Abeta generation.     

Tissue-specific expression and dimerization of the endoplasmic reticulum oxidoreductase Ero1beta

Endoplasmic reticulum oxidoreductases (Eros) are essential for the formation of disulfide bonds. Understanding disulfide bond catalysis in mammals is important because of the involvement of protein misfolding in conditions such as diabetes, arthritis, cancer, and aging. Mammals express two related Ero proteins, Ero1alpha and Ero1beta. Ero1beta is incompletely characterized but is of physiological interest because it is induced by the unfolded protein response. Here, we show that Ero1beta can form homodimers and mixed heterodimers with Ero1alpha, in addition to Ero-PDI dimers. Ero-Ero dimers require the Ero active site, occur in vivo, and can be modeled onto the Ero1p crystal structure. Our data indicate that the Ero1beta protein is constitutively strongly expressed in the stomach and the pancreas, but in a cell-specific fashion. In the stomach, selective expression of Ero1beta occurs in the enzyme-producing chief cells. In pancreatic islets, Ero1beta expression is high, but is inversely correlated with PDI and PDIp levels, demonstrating that cell-specific differences exist in the regulation of oxidative protein folding in vivo.     

Protein retention in yeast rough endoplasmic reticulum: expression and assembly of human ribophorin I

The RER retains a specific subset of ER proteins, many of which have been shown to participate in the translocation of nascent secretory and membrane proteins. The mechanism of retention of RER specific membrane proteins is unknown. To study this phenomenon in yeast, where no RER- specific membrane proteins have yet been identified, we expressed the human RER-specific protein, ribophorin I. In all mammalian cell types examined, ribophorin I has been shown to be restricted to the membrane of the RER. Here we ascertain that yeast cells correctly target, assemble, and retain ribophorin I in their RER. Floatation experiments demonstrated that human ribophorin I, expressed in yeast, was membrane associated. Carbonate (pH = 11) washing and Triton X-114 cloud-point precipitations of yeast microsomes indicated that ribophorin I was integrated into the membrane bilayer. Both chromatography on Con A and digestion with endoglycosidase H were used to prove that ribophorin I was glycosylated once, consistent with its expression in mammalian cells. Proteolysis of microsomal membranes and subsequent immunoblotting showed ribophorin I to have assumed the correct transmembrane topology. Sucrose gradient centrifugation studies found ribophorin I to be included only in fractions containing rough membranes and excluded from smooth ones that, on the basis of the distribution of BiP, included smooth ER. Ribosome removal from rough membranes and subsequent isopycnic centrifugation resulted in a shift in the buoyant density of the ribophorin I-containing membranes. Furthermore, the rough and density-shifted fractions were the exclusive location of protein translocation activity. Based on these studies we conclude that sequestration of membrane proteins to rough domains of ER probably occurs in a like manner in yeast and mammalian cells.     

Bax inhibitor-1 regulates endoplasmic reticulum stress-associated reactive oxygen species and heme oxygenase-1 expression

The Bax inhibitor-1 (BI-1) is an anti-apoptotic protein that is located in endoplasmic reticulum (ER) membranes and protects cells from ER stress-induced apoptosis. The ER is associated with generation of reactive oxygen species (ROS) through oxidative protein folding. This study examined the role of BI-1 in the regulation of ER stress-induced accumulation of ROS and expression of unfolded protein response-associated proteins. BI-1 reduced the expression levels of glucose response protein 78, C/EBP homologous protein, phospho-eukaryotic initiation factor 2alpha, IRE1alpha, XBP-1, and phospho-JNK and inhibited the cleavage of ATF-6alpha p-90, leading to the inhibition of ROS. Although ROS scavengers offer some protection against ER stress-induced apoptosis, the expression of pro-apoptotic ER stress proteins was not affected. This study shows that the response of unfolded proteins is followed by ROS accumulation under ER stress, which is regulated in BI-1 cells. The mechanism for these BI-1-associated functions involves the expression of heme oxygenase-1 (HO-1) through nuclear factor erythroid 2-related factor 2. In BI-1 cells, the transfection of HO-1 small interfering RNA completely abolished the BI-1-induced protection. The endogenous expression of HO-1 through ER stress-initiated ROS is believed to be as a protection signal. In conclusion, these observations suggest that BI-1 can inhibit the ER stress proteins as well as the accumulation of ROS, thereby protecting the cells. Moreover, HO-1 plays an important role in the BI-1-associated protection against ER stress.     

Folding of the human immunodeficiency virus type 1 envelope glycoprotein in the endoplasmic reticulum

The lumen of the endoplasmic reticulum (ER) provides a unique folding environment that is distinct from other organelles supporting protein folding. The relatively oxidizing milieu allows the formation of disulfide bonds. N-linked oligosaccharides that are attached during synthesis play multiple roles in the folding process of glycoproteins. They stabilize folded domains and increase protein solubility, which prevents aggregation of folding intermediates. Glycans mediate the interaction of newly synthesized glycoproteins with some resident ER folding factors, such as calnexin and calreticulin. Here we present an overview of the present knowledge on the folding process of the heavily glycosylated human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein in the ER.     

Distribution of the murine plasma cell antigen PC-1 in non-lymphoid tissues

PC-1 is an alloantigen of murine plasma cells. Its close association with secretory function in lymphoid cells previously raised the question of whether PC-1 was part of the secretory apparatus. In addition to its expression on lymphocytes, PC-1 had been known to be present in liver, brain, and kidney, although the data were derived almost entirely from bulk absorption studies of polyclonal alloantisera, and virtually nothing was known about the nature of the cells expressing PC-1 in these organs. If PC-1 was functionally involved in the secretory process. it might be expected to be present at secretory sites within these and other organs. We now report the results of an immunohistochemical survey of the distribution of PC-1 in a variety of non-lymphoid organs, using a mAb. The PC-1 Ag was found in a small number of highly discrete locations that were mostly, but not exclusively, associated with epithelia. Sites of strong expression included the distal convoluted tubule of the kidney, ducts of the salivary glands, epididymis, proximal part of the vas deferens, and chondrocytes. The PC-1 glycoprotein was also found in the capillaries of the brain, but did not appear to be present in capillaries elsewhere, a pattern that is strikingly similar to that of the receptor for the iron transport protein, transferrin. Negative sites included the thyroid, pancreas, choroid plexus, smooth and striated muscle, stomach, small and large intestine, gall bladder, renal glomeruli, testis, and seminal vesicles. These results are not consistent with a generalized role for PC-1 in secretion, but are compatible with a role in a specialized subset of macromolecular transport events.     

Mitochondria and endoplasmic reticulum: mitochondria-endoplasmic reticulum interplay in type 2 diabetes pathophysiology

Mitochondria and endoplasmic reticulum (ER) are two important metabolic organelles for the maintenance of cellular homeostasis and their functional defects are suspected to participate to the aetiology of type 2 diabetes (T2D). Particularly, excessive lipid intake and/or ectopic lipid accumulation in tissues (referred as lipotoxicity) are involved in alterations of both organelles and are closely linked to peripheral insulin resistance and defective insulin secretion. Since, mitochondria and ER are physically and functionally interconnected, their respective alterations during T2D could be interrelated. However, the mechanisms that coordinate the interplay between mitochondrial dysfunction and ER stress, and its relevance in the control of glucose homeostasis are unknown. Among these mechanisms, we will discuss on the potential role of altered mitochondria/ER crosstalk in organelle dysfunctions and in T2D pathophysiology.     

Bcl-2 and Ca2+ homeostasis in the endoplasmic reticulum

Recent data have revealed an unexpected role of Bcl-2 in modulating the steady-state levels and agonist-dependent fluxes of Ca(2+) ions. Direct monitoring of endoplasmic reticulum (ER) Ca(2+) concentration with recombinant probes reveals a lower state of filling in Bcl-2-overexpressing cells and a higher leak rate from the organelle. The broader set of indirect data using cytosolic probes reveals a more complex scenario, as in many cases no difference was detected in the Ca(2+) content of the intracellular pools. At the same time, Ca(2+) signals have been shown to affect important checkpoints of the apoptotic process, such as mitochondria, thus tuning the sensitivity of cells to various challenges. In this contribution, we will review (i) the data on the effect of Bcl-2 on [Ca(2+)](er), (ii) the functional significance of the Ca(2+)-signalling alteration and (iii) the current insight into the possible mechanisms of this effect.     

Downregulation of gap junction expression and function by endoplasmic reticulum stress

Gap junctional intercellular communication (GJIC) plays a critical role in the control of multiple cell behavior as well as in the maintenance of tissue and organ homeostasis. However, mechanisms involved in the regulation of gap junctions (GJs) have not been fully understood. Given endoplasmic reticulum (ER) stress and dysfunction of GJs coexist in several pathological situations, we asked whether GJs could be regulated by ER stress. Incubation of mesangial cells with ER stress‐inducing agents (thapsigargin, tunicamycin, and AB₅ subtilase cytotoxin) resulted in a decrease in connexin 43 (Cx43) expression at both protein and mRNA levels. This was accompanied by a loss of GJIC, as evidenced by the reduced numbers of dye‐coupled cells after single cell microinjection or scrape loading dye transfer. Further studies demonstrated that ER stress significantly inhibited the promoter activity of the Cx43 gene, reduced [³⁵S]‐methionine incorporation into Cx43 protein and accelerated degradation of Cx43. ER stress also decreased the Cx43 protein levels in several different cell types, including human umbilical vein endothelial cells, mouse‐derived renin‐secreting cells and human hepatoma cells. Furthermore, induction of ER stress by hypoxic chemicals thenoyltrifluoroacetone and cobalt chloride was found to be associated with a reduction in Cx43. Our findings thus reveal a close link between ER stress and GJs. ER stress may represent a novel mechanism underlying the altered GJs in a variety of pathological situations. J. Cell. Biochem. 107: 973–983, 2009. © 2009 Wiley‐Liss, Inc.     

Homocysteine-induced endoplasmic reticulum protein (Herp) is up-regulated in sporadic inclusion-body myositis and in endoplasmic reticulum stress-induced cultured human muscle fibers

Herp is a stress-response protein localized in the endoplasmic reticulum (ER) membrane. Herp was proposed to improve ER-folding, decrease ER protein load, and participate in ER-associated degradation (ERAD). Intra-muscle-fiber ubiquitinated multiprotein-aggregates containing, among other proteins, either amyloid-beta (Abeta) or phosphorylated tau are characteristic of sporadic inclusion-body myositis (s-IBM). ER stress and proteasome inhibition appear to play a role in s-IBM pathogenesis. We have now studied Herp in s-IBM muscle fibers and in ER-stress-induced or proteasome-inhibited cultured human muscle fibers. In s-IBM muscle fibers: (i) Herp was strongly immunoreactive in the form of aggregates, which co-localized with Abeta, GRP78, and beta2 proteasome subunit; (ii) Herp mRNA and protein were increased. In ER-stress-induced cultured human muscle fibers: (i) Herp immunoreactivity was diffusely increased; (ii) Herp mRNA and protein were increased. In proteasome-inhibited cultured human muscle fibers: (i) Herp immunoreactivity was in the form of aggregates; (ii) Herp protein was increased, but its mRNA was not. Accordingly, in s-IBM muscle fibers: (i) increase of Herp might be due to both ER-stress and proteasome inhibition; (ii) co-localization of Herp with Abeta, proteasome, and ER-chaperone GRP78 could reflect its possible role in processing and degradation of cytotoxic proteins in ER.