Efficient Endoplasmic Reticulum Research Articles

An anaplerotic approach to correct the mitochondrial dysfunction in ataxia-telangiectasia (A-T)

BackgroundATM, the protein defective in the human genetic disorder, ataxia-telangiectasia (A-T) plays a central role in response to DNA double-strand breaks (DSBs) and in protecting the cell against oxidative stress. We showed that A-T cells are hypersensitive to metabolic stress which can be accounted for by a failure to exhibit efficient endoplasmic reticulum (ER)-mitochondrial signalling and Ca2+ transfer in response to nutrient deprivation resulting in mitochondrial dysfunction. The objective of the current study is to use an anaplerotic approach using the fatty acid, heptanoate (C7), a metabolic product of the triglyceride, triheptanoin to correct the defect in ER-mitochondrial signalling and enhance cell survival of A-T cells in response to metabolic stress. MethodsWe treated control cells and A-T cells with the anaplerotic agent, heptanoate to determine their sensitivity to metabolic stress induced by inhibition of glycolysis with 2- deoxyglucose (2DG) using live-cell imaging to monitor cell survival for 72 h using the Incucyte system. We examined ER-mitochondrial signalling in A-T cells exposed to metabolic stress using a suite of techniques including immunofluorescence staining of Grp75, ER-mitochondrial Ca2+ channel, the VAPB-PTPIP51 ER-mitochondrial tether complexes as well as proximity ligation assays between Grp75-IP3R1 and VAPB1-PTPIP51 to establish a functional interaction between ER and mitochondria. Finally, we also performed metabolomic analysis using LC-MS/MS assay to determine altered levels of TCA intermediates A-T cells compared to healthy control cells. ResultsWe demonstrate that heptanoate corrects all aspects of the defective ER-mitochondrial signalling observed in A-T cells. Heptanoate enhances ER-mitochondrial contacts; increases the flow of calcium from the ER to the mitochondrion; restores normal mitochondrial function and mitophagy and increases the resistance of ATM-deficient cells and cells from A-T patients to metabolic stress-induced killing. The defect in mitochondrial function in ATM-deficient cells was accompanied by more reliance on aerobic glycolysis as shown by increased lactate dehydrogenase A (LDHA), accumulation of lactate, and reduced levels of both acetyl CoA and ATP which are all restored by heptanoate. ConclusionsWe conclude that heptanoate corrects metabolic stress in A-T cells by restoring ER-mitochondria signalling and mitochondrial function and suggest that the parent compound, triheptanoin, has immense potential as a novel therapeutic agent for patients with A-T.

Cdc1 removes the ethanolamine phosphate of the first mannose of GPI anchors and thereby facilitates the integration of GPI proteins into the yeast cell wall.

Temperature-sensitive cdc1(ts) mutants are reported to stop the cell cycle upon a shift to 30°C in early G2, that is, as small budded cells having completed DNA replication but unable to duplicate the spindle pole body. A recent report showed that PGAP5, a human homologue of CDC1, acts as a phosphodiesterase removing an ethanolamine phosphate (EtN-P) from mannose 2 of the glycosylphosphatidylinositol (GPI) anchor, thus permitting efficient endoplasmic reticulum (ER)-to-Golgi transport of GPI proteins. We find that the essential CDC1 gene can be deleted in mcd4∆ cells, which do not attach EtN-P to mannose 1 of the GPI anchor, suggesting that Cdc1 removes the EtN-P added by Mcd4. Cdc1-314(ts) mutants do not accumulate GPI proteins in the ER but have a partial secretion block later in the secretory pathway. Growth tests and the genetic interaction profile of cdc1-314(ts) pinpoint a distinct cell wall defect. Osmotic support restores GPI protein secretion and actin polarization but not growth. Cell walls of cdc1-314(ts) mutants contain large amounts of GPI proteins that are easily released by β-glucanases and not attached to cell wall β1,6-glucans and that retain their original GPI anchor lipid. This suggests that the presumed transglycosidases Dfg5 and Dcw1 of cdc1-314(ts) transfer GPI proteins to cell wall β1,6-glucans inefficiently.

The endoplasmic reticulum stress marker CHOP predicts survival in malignant mesothelioma

Background:Mesothelioma is an incurable cancer originating from the mesothelial cells that line the pleural, peritoneal and pericardial cavities. These cells synthesise large quantities of surface glycoproteins, rendering them dependent upon efficient endoplasmic reticulum (ER) function. When faced with elevated levels of secretory protein load, cells are said to experience ER stress, which has been implicated in the pathogenesis of many human diseases including cancer.Method:We set out to measure markers of ER stress in malignant mesothelioma and to determine whether ER stress signalling correlates with clinical parameters.Results:We observed that expression of the ER stress-responsive transcription factor C/EBP homologous protein (CHOP) correlated with patient survival and remained an independent prognostic variable in pairwise comparisons with all clinical variables tested. The most parsimonious multivariate model in our study comprised only performance status and CHOP staining. In contrast, expression of the ER stress-responsive phosphatase growth arrest and DNA damage 34 (GADD34) correlated with the degree of mesothelial differentiation, being lost progressively in biphasic and sarcomatoid mesotheliomas.Conclusion:Our findings suggest that staining for CHOP provides prognostic information that may be useful in the stratification of patients with mesothelioma. Staining for GADD34 may prove useful in classification of mesothelioma histopathology.

A KDEL‐tagged monoclonal antibody is efficiently retained in the endoplasmic reticulum in leaves, but is both partially secreted and sorted to protein storage vacuoles in seeds

Transgenic plants are attractive biological systems for the large-scale production of pharmaceutical proteins. In particular, seeds offer special advantages, such as ease of handling and long-term stable storage. Nevertheless, most of the studies of the expression of antibodies in plants have been performed in leaves. We report the expression of a secreted (sec-Ab) or KDEL-tagged (Ab-KDEL) mutant of the 14D9 monoclonal antibody in transgenic tobacco leaves and seeds. Although the KDEL sequence has little effect on the accumulation of the antibody in leaves, it leads to a higher antibody yield in seeds. sec-Ab(Leaf) purified from leaf contains complex N-glycans, including Lewis(a) epitopes, as typically found in extracellular glycoproteins. In contrast, Ab-KDEL(Leaf) bears only high-mannose-type oligosaccharides (mostly Man 7 and 8) consistent with an efficient endoplasmic reticulum (ER) retention/cis-Golgi retrieval of the antibody. sec-Ab and Ab-KDEL gamma chains purified from seeds are cleaved by proteases and contain complex N-glycans indicating maturation in the late Golgi compartments. Consistent with glycosylation of the protein, Ab-KDEL(Seed) was partially secreted and sorted to protein storage vacuoles (PSVs) in seeds and not found in the ER. This dual targeting may be due to KDEL-mediated targeting to the PSV and to a partial saturation of the vacuolar sorting machinery. Taken together, our results reveal important differences in the ER retention and vacuolar sorting machinery between leaves and seeds. In addition, we demonstrate that a plant-made antibody with triantennary high-mannose-type N-glycans has similar Fab functionality to its counterpart with biantennary complex N-glycans, but the former antibody interacts with protein A in a stronger manner and is more immunogenic than the latter. Such differences could be related to a variable immunoglobulin G (IgG)-Fc folding that would depend on the size of the N-glycan.

PH-induced Conversion of the Transport Lectin ERGIC-53 Triggers Glycoprotein Release

The recycling mannose lectin ERGIC-53 operates as a transport receptor by mediating efficient endoplasmic reticulum (ER) export of some secretory glycoproteins. Binding of cargo to ERGIC-53 in the ER requires Ca2+. Cargo release occurs in the ERGIC, but the molecular mechanism is unknown. Here we report efficient binding of purified ERGIC-53 to immobilized mannose at pH 7.4, the pH of the ER, but not at slightly lower pH. pH sensitivity of the lectin was more prominent when Ca2+ concentrations were low. A conserved histidine in the center of the carbohydrate recognition domain was required for lectin activity suggesting it may serve as a molecular pH/Ca2+ sensor. Acidification of cells inhibited the association of ERGIC-53 with the known cargo cathepsin Z-related protein and dissociation of this glycoprotein in the ERGIC was impaired by organelle neutralization that did not impair the transport of a control protein. The results elucidate the molecular mechanism underlying reversible lectin/cargo interaction and establish the ERGIC as the earliest low pH site of the secretory pathway.