Regulator of G Signaling 16 is a marker for the distinct endoplasmic reticulum stress state associated with aggregated mutant alpha1-antitrypsin Z in the classical form of alpha1-antitrypsin deficiency

In the classical form of alpha(1)-antitrypsin deficiency, a mutant protein accumulates in a polymerized form in the endoplasmic reticulum (ER) of liver cells causing liver damage and carcinogenesis by a gain-of-toxic function mechanism. Recent studies have indicated that the accumulation of mutant alpha(1)-antitrypsin Z in the ER specifically activates the autophagic response but not the unfolded protein response and that autophagy plays a critical role in disposal of insoluble alpha(1)-antitrypsin Z. In this study, we used genomic analysis of the liver in a novel transgenic mouse model with inducible expression to screen for changes in gene expression that would potentially define how the liver responds to accumulation of this mutant protein. There was no unfolded protein response. Of several distinct gene expression profiles, marked up-regulation of regulator of G signaling (RGS16) was particularly notable. RGS16 did not increase when model systems were exposed to classical inducers of ER stress, including tunicamycin and calcium ionophore, or when a nonpolymerogenic alpha(1)-antitrypsin mutant accumulated in the ER. RGS16 was up-regulated in livers from patients with alpha(1)-antitrypsin deficiency, and the degree of up-regulation correlated with the hepatic levels of insoluble alpha(1)-antitrypsin Z protein. Taken together, these results indicate that expression of RGS16 is an excellent marker for the distinct form of "ER stress" that occurs in alpha(1)-antitrypsin deficiency, presumably determined by the aggregation-prone properties of the mutant protein that characterizes the deficiency.     

Selenoprotein S/SEPS1 Modifies Endoplasmic Reticulum Stress in Z Variant ��1-Antitrypsin Deficiency

Z α₁-antitrypsin (ZAAT) deficiency is a disease associated with emphysematous lung disease and also with liver disease. The liver disease of AAT deficiency is associated with endoplasmic reticulum (ER) stress. SEPS1 is a selenoprotein that, through a chaperone activity, decreases ER stress. To determine the effect of SEPS1 on ER stress in ZAAT deficiency, we measured activity of the grp78 promoter and levels of active ATF6 as markers of the unfolded protein response in HepG2 cells transfected with the mutant form of AAT, a ZAAT transgene. We evaluated levels of NFκB activity as a marker of the ER overload response. To determine the effect of selenium supplementation on the function of SEPS1, we investigated glutathione peroxidase activity, grp78 promoter activity, and NFκB activity in the presence or absence of selenium. SEPS1 reduced levels of active ATF6. Overexpression of SEPS1 also inhibited grp78 promoter and NFκB activity, and this effect was enhanced in the presence of selenium supplementation. This finding demonstrates a role for SEPS1 in ZAAT deficiency and suggests a possible therapeutic potential for selenium supplementation.SEPS1 (selenoprotein S, VIMP, Tanis, or SelS) is a selenoprotein found in the endoplasmic reticulum (ER)³ membrane. SEPS1 participates in the processing and removal of misfolded proteins from the ER to the cytosol, where they are polyubiquitinated and degraded through the proteasome (1). SEPS1 can be induced by ER stress (2) and has been shown in macrophages to be protective from pharmacological ER stress agent-induced apoptosis (3).The endoplasmic reticulum is one of the largest cell organelles. It serves many essential functions, including production of all components of cellular membranes, proteins, lipids, and sterols (4). Only correctly folded proteins are transported out of the ER, whereas incompletely folded proteins are retained in the organelle to complete the folding process or be targeted for destruction. ER stress is defined as an imbalance between the cellular demand for ER function and capacity of the organelle. It is characterized by a number of intracellular responses. These responses include the ER overload response (EOR), the unfolded protein response (UPR), and apoptosis.α₁-Antitrypsin (AAT) deficiency is a disease characterized by early onset emphysema and liver disease (5). The mutant Z form of this autosomal co-dominant disease occurs in >95% of all individuals with AAT deficiency (6). Liver disease occurs in ∼10% of all homozygous neonates who develop hepatitis and cholestasis. A proportion of these children progress to liver failure, requiring liver transplantation (7, 8). Cirrhosis can also occur in adults without a preceding history of childhood liver disease. The mutant Z AAT polymerizes and accumulates in the ER, leaving only 15% of ZAAT secreted (9, 10). This accumulation of abnormal protein in the ER gives rise to ER stress, which is believed to contribute to the liver disease that results from AAT deficiency. The cells respond to this perturbation by inducing the expression of novel genes whose products aim to restore normal ER function (11). SEPS1 is an example of a molecular chaperone that serves to augment the capacity of the ER for protein folding and degradation.In this paper, we investigate the role of SEPS1 in regulating the cellular response to ER stress in HepG2 cells transfected with the ZAAT transgene. We investigate the effect of SEPS1 on the UPR component of this response by measuring grp78 promoter activity, a UPR-up-regulated gene that functions as a molecular chaperone, and by detecting activated ATF6, which occurs downstream to the activation of grp78. The EOR component of ER stress is investigated by measuring NFκB activation.We study the effect of selenium supplementation on the action of SEPS1 to see if the function of this selenoprotein can be enhanced and, if so, through which pathways, looking specifically at grp78 promoter activity, ATF6 activation, and NFκB activation and also at glutathione peroxidase (GPx) activity and at the anti-inflammatory 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) pathway. GPx is a selenoprotein whose activity can be readily assayed. This was used as a measure of selenoprotein activity.The role of SEPS1 in conformational diseases has not been evaluated. These diseases are caused by inherited or acquired modifications in protein structure, where specific proteins undergo a conformational rearrangement, causing deposition within cellular compartments, such as the ER. This can lead to devastating results. AAT deficiency is one such disease, but the group includes Alzheimer, Parkinson, and Huntington diseases and cystic fibrosis.     

The impact of the unfolded protein response on human disease

A central function of the endoplasmic reticulum (ER) is to coordinate protein biosynthetic and secretory activities in the cell. Alterations in ER homeostasis cause accumulation of misfolded/unfolded proteins in the ER. To maintain ER homeostasis, eukaryotic cells have evolved the unfolded protein response (UPR), an essential adaptive intracellular signaling pathway that responds to metabolic, oxidative stress, and inflammatory response pathways. The UPR has been implicated in a variety of diseases including metabolic disease, neurodegenerative disease, inflammatory disease, and cancer. Signaling components of the UPR are emerging as potential targets for intervention and treatment of human disease.     

Endoplasmic reticulum: the unfolded protein response is tangled in neurodegeneration

The endoplasmic reticulum (ER) is involved in the folding and maturation of membrane-bound and secreted proteins. Disturbed homeostasis in the ER can lead to accumulation of misfolded proteins, which trigger a stress response called the unfolded protein response (UPR). In neurodegenerative diseases that are classified as tauopathies, activation of the UPR coincides with the pathogenic accumulation of the microtubule associated protein tau. Several lines of evidence indicate that UPR activation contributes to increased levels of phosphorylated tau, a prerequisite for the formation of tau aggregates. Increased understanding of the crosstalk between signaling pathways involved in protein quality control in the ER and tau phosphorylation will support the development of new therapeutic targets that promote neuronal survival.     

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.     

Retention of mutant alpha(1)-antitrypsin Z in endoplasmic reticulum is associated with an autophagic response

Although there is evidence for specific subcellular morphological alterations in response to accumulation of misfolded proteins in the endoplasmic reticulum (ER), it is not clear whether these morphological changes are stereotypical or if they depend on the specific misfolded protein retained. This issue may be particularly important for mutant secretory protein alpha(1)-antitrypsin (alpha(1)AT) Z because retention of this mutant protein in the ER can cause severe target organ injury, the chronic hepatitis/hepatocellular carcinoma associated with alpha(1)AT deficiency. Here we examined the morphological changes that occur in human fibroblasts engineered for expression and ER retention of mutant alpha(1)ATZ and in human liver from three alpha(1)AT-deficient patients. In addition to marked expansion and dilatation of ER, there was an intense autophagic response. Mutant alpha(1)ATZ molecules were detected in autophagosomes by immune electron microscopy, and intracellular degradation of alpha(1)ATZ was partially reduced by chemical inhibitors of autophagy. In contrast to mutant CFTRDeltaF508, expression of mutant alpha(1)ATZ in heterologous cells did not result in the formation of aggresomes. These results show that ER retention of mutant alpha(1)ATZ is associated with a marked autophagic response and raise the possibility that autophagy represents a mechanism by which liver of alpha(1)AT-deficient patients attempts to protect itself from injury and carcinogenesis.     

Enhanced ubiquitin-proteasome activity in calreticulin deficient cells: a compensatory mechanism for cell survival

Calreticulin is a lectin chaperone essential for intracellular calcium homeostasis. Deletion of calreticulin gene compromises the overall quality control within the endoplasmic reticulum (ER) leading to activation of the unfolded protein response. However, the ER structure of calreticulin deficient cells (crt-/-) is not altered due to accumulation of misfolded proteins. Therefore, the aim of this study was to determine whether the ubiquitin-proteasome pathway is activated in crt-/- cells as a compensatory mechanism for cell survival. Here we show a significant increase in the expression of genes involved in ER associated degradation and activation of the ubiquitin-proteasome system in crt-/- cells. We also demonstrated that the ubiquitination of two proteins processed in ER, connexin 43 and A1AT NHK (alpha1-antitrypsin mutant) are increased in crt-/- cells. Furthermore, we showed that the increased proteasome activity in the crt-/- cells could be rescued upon re-introduction of calreticulin or calsequestrin (a muscle calcium binding protein). We also illustrated that increased cytosolic Ca2+ enhances the proteasome activity. Interestingly, suppression of calnexin function using siRNA further elevated the proteasome activity in crt-/- cells. This is the first report to show that loss of calreticulin function enhances the ubiquitin-proteasome activity which could function as a compensatory mechanism for cell survival.     

ER stress protects from retinal degeneration

The unfolded protein response (UPR) is a specific cellular process that allows the cell to cope with the overload of unfolded/misfolded proteins in the endoplasmic reticulum (ER). ER stress is commonly associated with degenerative pathologies, but its role in disease progression is still a matter for debate. Here, we found that mutations in the ER‐resident chaperone, neither inactivation nor afterpotential A (NinaA), lead to mild ER stress, protecting photoreceptor neurons from various death stimuli in adult Drosophila. In addition, Drosophila S2 cultured cells, when pre‐exposed to mild ER stress, are protected from H₂O₂, cycloheximide‐ or ultraviolet‐induced cell death. We show that a specific ER‐mediated signal promotes antioxidant defences and inhibits caspase‐dependent cell death. We propose that an immediate consequence of the UPR not only limits the accumulation of misfolded proteins but also protects tissues from harmful exogenous stresses.     

Lipid-Induced ER Stress: Synergistic Effects of Sterols and Saturated Fatty Acids

Stress within the endoplasmic reticulum (ER) induces a coordinated response, namely the unfolded protein response (UPR), devoted to helping the ER cope with the accumulation of misfolded proteins. Failure of the UPR plays an important role in several human diseases. Recent studies report that intracellular accumulation of saturated fatty acids (SFAs) and cholesterol, seen in diseases of high incidence, such as obesity or atherosclerosis, results in ER stress. In the present study, we evaluated the effects of perturbations to lipid homeostasis on ER stress/UPR induction in the model eukaryote Saccharomyces cerevisiae . We show that SFA originating from either endogenous (preclusion of fatty acid desaturation) or exogenous (feeding with extracellular SFA) sources trigger ER stress and that ergosterol, the major sterol in yeast, acts synergistically with SFA in this process. This latter effect is connected to ergosterol accumulation within microsomal fractions from SFA-accumulating cells, which display highly saturated phospholipid content. Moreover, treating the cells with the molecular chaperone 4-phenyl butyrate abolishes UPR induction, suggesting that lipid-induced ER stress leads to an overload of misfolded protein that acts, in turn, as the molecular signal for induction of the UPR. The present data are discussed in the context of human diseases that involve lipid deregulation.     

The unfolded protein response in hereditary haemochromatosis

To cope with the accumulation of unfolded or misfolded proteins the endoplasmic reticulum (ER) has evolved specific signalling pathways collectively called the unfolded protein response (UPR). Elucidation of the mechanisms governing ER stress signalling has linked this response to the regulation of diverse physiologic processes as well as to the progression of a number of diseases. Interest in hereditary haemochromatosis (HH) has focused on the study of proteins implicated in iron homeostasis and on the identification of new alleles related with the disease. HFE has been amongst the preferred targets of interest, since the discovery that its C282Y mutation was associated with HH. However, the discrepancies between the disease penetrance and the frequency of this mutation have raised the possibility that its contribution to disease progression might go beyond the mere involvement in regulation of cellular iron uptake. Recent findings revealed that activation of the UPR is a feature of HH and that this stress response may be involved in the genesis of immunological anomalies associated with the disease. This review addresses the connection of the UPR with HH, including its role in MHC-I antigen presentation pathway and possible implications for new clinical approaches to HH.     

Accumulation of mutant alpha1-antitrypsin Z in the endoplasmic reticulum activates caspases-4 and -12, NFkappaB, and BAP31 but not the unfolded protein response

In alpha(1)-antitrypsin (alpha1AT) deficiency, a polymerogenic mutant form of the secretory glycoprotein alpha1AT, alpha1ATZ, is retained in the endoplasmic reticulum (ER) of liver cells. It is not yet known how this results in liver injury in a subgroup of deficient individuals and how the remainder of deficient individuals escapes liver disease. One possible explanation is that the "susceptible" subgroup is unable to mount the appropriate protective cellular responses. Here we examined the effect of mutant alpha1ATZ on several potential protective signaling pathways by using cell lines with inducible expression of mutant alpha1AT as well as liver from transgenic mice with liver-specific inducible expression of mutant alpha1AT. The results show that ER retention of polymerogenic mutant alpha1ATZ does not result in an unfolded protein response (UPR). The UPR can be induced in the presence of alpha1ATZ by tunicamycin excluding the possibility that the pathway has been disabled. In striking contrast, ER retention of nonpolymerogenic alpha1AT mutants does induce the UPR. These results indicate that the machinery responsible for activation of the UPR can distinguish the physical characteristics of proteins that accumulate in the ER in such a way that it can respond to misfolded but not relatively ordered polymeric structures. Accumulation of mutant alpha1ATZ does activate specific signaling pathways, including caspase-12 in mouse, caspase-4 in human, NFkappaB, and BAP31, a profile that was distinct from that activated by nonpolymerogenic alpha1AT mutants.     

Ligand-independent dimerization activates the stress response kinases IRE1 and PERK in the lumen of the endoplasmic reticulum

IRE1 and PERK are type I transmembrane serine/threonine protein kinases that are activated by unfolded proteins in the endoplasmic reticulum (ER) to signal adaptive responses. IRE1 is present in all eukaryotic cells and signals the unfolded protein response through its kinase and endoribonuclease activities. PERK signals phosphorylation of a translation initiation factor to inhibit protein synthesis in higher eukaryotic cells but is absent in the Saccharomyces cerevisiae genome. The amino acid sequences of the amino-terminal ER luminal domains (NLDs) from IRE1 and PERK display limited homology and have diverged among species. In this study, we have demonstrated that the NLD of yeast Ire1p is required for signaling. However, the NLDs from human IRE1alpha and murine IRE1beta and the Caenorhabditis elegans IRE1 and PERK function as replacements for the S. cerevisiae Ire1p-NLD to signal the unfolded protein response. Replacement of the Ire1p-NLD with a functional leucine zipper dimerization motif yielded a constitutively active kinase that surprisingly was further activated by ER stress. These results demonstrate that ER stress-induced dimerization of the NLD is sufficient for IRE1 and PERK activation and is conserved through evolution. We propose that ligand-independent activation of IRE1 and PERK permits homodimerization upon accumulation of unfolded proteins in the lumen of the ER.