FAD-linked presenilin-1 mutants impede translation regulation under ER stress

FAD mutations in presenilin-1 (PS1) cause attenuation of the induction of the endoplasmic reticulum (ER)-resident chaperone GRP78/BiP under ER stress, due to disturbed function of IRE1, the sensor for accumulation of unfolded protein in the ER lumen. PERK, an ER-resident transmembrane protein kinase, is also a sensor for the unfolded protein response (UPR), causing phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) to inhibit translation initiation. Here, we report that the FAD mutant PS1 disturbs the UPR by attenuating both the activation of PERK and the phosphorylation of eIF2alpha. Consistent with the results of a disturbed UPR, inhibition of protein synthesis under ER stress was impaired in cells expressing PS1 mutants. These results suggest that mutant PS1 impedes general translational attenuation regulated by PERK and eIF2alpha, resulting in an increased load of newly synthesized proteins into the ER and subsequently increasing vulnerability to ER stress.     

Endoplasmic reticulum stress response in yeast and humans

Stress pathways monitor intracellular systems and deploy a range of regulatory mechanisms in response to stress. One of the best-characterized pathways, the UPR (unfolded protein response), is an intracellular signal transduction pathway that monitors ER (endoplasmic reticulum) homoeostasis. Its activation is required to alleviate the effects of ER stress and is highly conserved from yeast to human. Although metazoans have three UPR outputs, yeast cells rely exclusively on the Ire1 (inositol-requiring enzyme-1) pathway, which is conserved in all Eukaryotes. In general, the UPR program activates hundreds of genes to alleviate ER stress but it can lead to apoptosis if the system fails to restore homoeostasis. In this review, we summarize the major advances in understanding the response to ER stress in Sc (Saccharomyces cerevisiae), Sp (Schizosaccharomyces pombe) and humans. The contribution of solved protein structures to a better understanding of the UPR pathway is discussed. Finally, we cover the interplay of ER stress in the development of diseases.     

Hepatic Xbp1 Gene Deletion Promotes Endoplasmic Reticulum Stress-induced Liver Injury and Apoptosis

Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), a highly conserved signaling cascade that functions to alleviate stress and promote cell survival. If, however, the cell is unable to adapt and restore homeostasis, then the UPR activates pathways that promote apoptotic cell death. The molecular mechanisms governing the critical transition from adaptation and survival to initiation of apoptosis remain poorly understood. We aim to determine the role of hepatic Xbp1, a key mediator of the UPR, in controlling the adaptive response to ER stress in the liver. Liver-specific Xbp1 knockout mice (Xbp1^LKO) and Xbp1^fl/fl control mice were subjected to varying levels and durations of pharmacologic ER stress. Xbp1^LKO and Xbp1^fl/fl mice showed robust and equal activation of the UPR acutely after induction of ER stress. By 24 h, Xbp1^fl/fl controls showed complete resolution of UPR activation and no liver injury, indicating successful adaptation to the stress. Conversely, Xbp1^LKO mice showed ongoing UPR activation associated with progressive liver injury, apoptosis, and, ultimately, fibrosis by day 7 after induction of ER stress. These data indicate that hepatic XBP1 controls the adaptive response of the UPR and is critical to restoring homeostasis in the liver in response to ER stress.     

Gene expression profiling of yeasts overexpressing wild type or misfolded Pma1 variants reveals activation of the Hog1 MAPK pathway

Dominant negative PMA1 mutants render misfolded proteins that are retained in the endoplasmic reticulum (ER) and slowly degraded by ER-associated degradation. Accumulation of misfolded proteins in the ER activates an ER-to-nucleus signalling pathway termed the unfolded protein response (UPR). We have used a PMA1-D378T dominant negative mutant to analyse its impact on UPR induction. Our results show that overexpression of the misfolded mutant Pma1 does not lead to activation of the UPR. In addition, in mutants with a constitutively activated UPR the turnover rate of the mutant ATPase is not altered. To determine if the expression of the misfolded mutant protein induces some other kind of response we performed global gene expression analysis experiments in yeasts overexpressing either wild type or dominant lethal PMA1 alleles. The results suggest that the high osmolarity glycerol (Hog1) mitogen-activated protein kinase (MAPK) pathway is activated by both wild type and mutant ATPases. We show that expression of the PMA1 alleles induces phosphorylation of Hog1 and activation of the Hog1 MAPK cascade. This activation is mediated by the Sln1 branch of the stress-dependent Hog1 MAPK network. Finally, we show that at least two other plasma membrane proteins are also able to activate the Hog1 MAPK system.     

Tumor necrosis factor alpha (TNFalpha) induces the unfolded protein response (UPR) in a reactive oxygen species (ROS)-dependent fashion, and the UPR counteracts ROS accumulation by TNFalpha

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER overload, resulting in ER stress. To cope with ER stress, mammalian cells trigger a specific response known as the unfolded protein response (UPR). Although recent studies have indicated cross-talk between ER stress and oxidative stress, the mechanistic link is not fully understood. By using murine fibrosarcoma L929 cells, in which tumor necrosis factor (TNF) alpha induces accumulation of reactive oxygen species (ROS) and cell death, we show that TNFalpha induces the UPR in a ROS-dependent fashion. In contrast to TNFalpha, oxidative stresses by H2O2 or arsenite only induce eukaroytic initiation factor 2alpha phosphorylation, but not activation of PERK- or IRE1-dependent pathways, indicating the specificity of downstream signaling induced by various oxidative stresses. Conversely, the UPR induced by tunicamycin substantially suppresses TNFalpha-induced ROS accumulation and cell death by inhibiting reduction of cellular glutathione levels. Collectively, some, but not all, oxidative stresses induce the UPR, and pre-emptive UPR counteracts TNFalpha-induced ROS accumulation.     

Integrating stress signals at the endoplasmic reticulum: The BCL-2 protein family rheostat

The assembling of distinct signaling protein complexes at the endoplasmic reticulum (ER) membrane controls several stress responses related to calcium homeostasis, autophagy, ER morphogenesis and protein folding. Diverse pathological conditions interfere with the function of the ER altering protein folding, a condition known as “ER stress”. Adaptation to ER stress depends on the activation of the unfolded protein response (UPR) and protein degradation pathways such as autophagy. Under chronic or irreversible ER stress, cells undergo apoptosis, where the BCL-2 protein family plays a crucial role at the mitochondria to trigger cytochrome c release and apoptosome assembly. Several BCL2 family members also regulate physiological processes at the ER through dynamic interactomes. Here we provide a comprehensive view of the roles of the BCL-2 family of proteins in mediating the molecular crosstalk between the ER and mitochondria to initiate apoptosis, in addition to their emerging functions in adaptation to stress, including autophagy, UPR, calcium homeostasis and organelle morphogenesis. We envision a model where BCL-2-containing complexes may operate as stress rheostats that, beyond their known apoptosis functions at the mitochondria, determine the amplitude and kinetics of adaptive responses against ER-related injuries. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

Unfolded Protein Response Signaling and Metabolic Diseases

The endoplasmic reticulum (ER) is a central organelle for protein biosynthesis, folding, and traffic. Perturbations in ER homeostasis create a condition termed ER stress and lead to activation of the complex signaling cascade called the unfolded protein response (UPR). Recent studies have documented that the UPR coordinates multiple signaling pathways and controls various physiologies in cells and the whole organism. Furthermore, unresolved ER stress has been implicated in a variety of metabolic disorders, such as obesity and type 2 diabetes. Therefore, intervening in ER stress and modulating signaling components of the UPR would provide promising therapeutics for the treatment of human metabolic diseases.     

The unfolded protein response in melanocytes: activation in response to chemical stressors of the endoplasmic reticulum and tyrosinase misfolding

Accumulation of proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), comprising three signaling pathways initiated by Ire1, Perk and Atf6 respectively. Unfolded protein response activation was compared in chemically stressed murine wildtype melanocytes and mutant melanocytes that retain tyrosinase in the ER. Thapsigargin, an ER stressor, activated all pathways in wildtype melanocytes, triggering Caspase 12-mediated apoptosis at toxic doses. Albino melanocytes expressing mutant tyrosinase showed evidence of ER stress with increased Ire1 expression, but the downstream effector, Xbp1, was not activated even following thapsigargin treatment. Attenuation of Ire1 signaling was recapitulated in wildtype melanocytes treated with thapsigargin for 8 days, with diminished Xbp1 activation observed after 4 days. Atf6 was also activated in albino melanocytes, with no response to thapsigargin, while the Perk pathway was not activated and thapsigargin treatment elicited robust expression of the downstream effector CCAAT-enhancer-binding protein homologous protein. Thus, melanocytes adapt to ER stress by attenuating two UPR pathways.     

Disruption of the ribosomal P complex leads to stress-induced autophagy

The human ribosomal P complex, which consists of the acidic ribosomal P proteins RPLP0, RPLP1, and RPLP2 (RPLP proteins), recruits translational factors, facilitating protein synthesis. Recently, we showed that overexpression of RPLP1 immortalizes primary cells and contributes to transformation. Moreover, RPLP proteins are overexpressed in human cancer, with the highest incidence in breast carcinomas. It is thought that disruption of the P complex would directly affect protein synthesis, causing cell growth arrest and eventually apoptosis. Here, we report a distinct mechanism by which cancer cells undergo cell cycle arrest and induced autophagy when RPLP proteins are downregulated. We found that absence of RPLP0, RPLP1, or RPLP2 resulted in reactive oxygen species (ROS) accumulation and MAPK1/ERK2 signaling pathway activation. Moreover, ROS generation led to endoplasmic reticulum (ER) stress that involved the EIF2AK3/PERK-EIF2S1/eIF2α-EIF2S2-EIF2S3-ATF4/ATF-4- and ATF6/ATF-6-dependent arms of the unfolded protein response (UPR). RPLP protein-deficient cells treated with autophagy inhibitors experienced apoptotic cell death as an alternative to autophagy. Strikingly, antioxidant treatment prevented UPR activation and autophagy while restoring the proliferative capacity of these cells. Our results indicate that ROS are a critical signal generated by disruption of the P complex that causes a cellular response that follows a sequential order: first ROS, then ER stress/UPR activation, and finally autophagy. Importantly, inhibition of the first step alone is able to restore the proliferative capacity of the cells, preventing UPR activation and autophagy. Overall, our results support a role for autophagy as a survival mechanism in response to stress due to RPLP protein deficiency.