Anti-ATF6 alpha Antibody [1-7] (A443)

ATF6α, a membrane-anchored transcription factor from the endoplasmic reticulum (ER) that modulates the cellular response to stress as an effector of the unfolded-protein response (UPR), is a key player in the development of tumors of different origin. ATF6α activation has been linked to oncogenic transformation and tumor maintenance; however, the mechanism(s) underlying this phenomenon remains elusive. Here, using a phenotypic small interfering RNA (siRNA) screening, we identified a novel role for ATF6α in chemoresistance and defined the protein disulfide isomerase A5 (PDIA5) as necessary for ATF6α activation upon ER stress. PDIA5 contributed to disulfide bond rearrangement in ATF6α under stress conditions, thereby leading to ATF6α export from the ER and activation of its target genes. Further analysis of the mechanism demonstrated that PDIA5 promotes ATF6α packaging into coat protein complex II (COPII) vesicles and that the PDIA5/ATF6α activation loop is essential to confer chemoresistance on cancer cells. Genetic and pharmacological inhibition of the PDIA5/ATF6α axis restored sensitivity to the drug treatment. This work defines the mechanisms underlying the role of ATF6α activation in carcinogenesis and chemoresistance; furthermore, it identifies PDIA5 as a key regulator ATF6α-mediated cellular functions in cancer.

The unfolded protein response (UPR) maintains endoplasmic reticulum (ER) proteostasis through the activation of transcription factors such as XBP1s and ATF6. The functional consequences of these transcription factors for ER proteostasis remain poorly defined. Here, we describe methodology that enables orthogonal, small-molecule-mediated activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the same cell independent of stress. We employ transcriptomics and quantitative proteomics to evaluate ER proteostasis network remodeling owing to the XBP1s and/or ATF6 transcriptional programs. Furthermore, we demonstrate that the three ER proteostasis environments accessible by activating XBP1s and/or ATF6 differentially influence the folding, trafficking, and degradation of destabilized ER client proteins without globally affecting the endogenous proteome. Our data reveal how the ER proteostasis network is remodeled by the XBP1s and/or ATF6 transcriptional programs at the molecular level and demonstrate the potential for selective restoration of aberrant ER proteostasis of pathologic, destabilized proteins through arm-selective UPR activation.

Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Endoplasmic reticulum (ER) stress transducers transduce signals from the ER to the cytoplasm and nucleus when unfolded proteins accumulate in the ER. BBF2 human homolog on chromosome 7 (BBF2H7) and old astrocyte specifically induced substance (OASIS), ER-resident transmembrane proteins, have recently been identified as novel ER stress transducers that have roles in chondrogenesis and osteogenesis, respectively. However, the molecular mechanisms that regulate the activation of BBF2H7 and OASIS under ER stress conditions remain unresolved. Here, we showed that BBF2H7 and OASIS are notably unstable proteins that are easily degraded via the ubiquitin-proteasome pathway under normal conditions. ER stress conditions enhanced the stability of BBF2H7 and OASIS, and promoted transcription of their target genes. HMG-CoA reductase degradation 1 (HRD1), an ER-resident E3 ubiquitin ligase, ubiquitinated BBF2H7 and OASIS under normal conditions, whereas ER stress conditions dissociated the interaction between HRD1 and BBF2H7 or OASIS. The stabilization of OASIS in Hrd1(-/-) cells enhanced the expression of collagen fibers during osteoblast differentiation, whereas a knockdown of OASIS in Hrd1(-/-) cells suppressed the production of collagen fibers. These findings suggest that ER stress stabilizes OASIS family members and this is a novel molecular mechanism for the activation of ER stress transducers.

The protein-disulfide isomerase (PDI) family member anterior gradient 2 (AGR2) is reportedly overexpressed in numerous cancers and plays a role in cancer development. However, to date the molecular functions of AGR2 remain to be characterized. Herein we have identified AGR2 as bound to newly synthesized cargo proteins using a proteomics analysis of endoplasmic reticulum (ER) membrane-bound ribosomes. Nascent protein chains that translocate into the ER associate with specific ER luminal proteins, which in turn ensures proper folding and posttranslational modifications. Using both imaging and biochemical approaches, we confirmed that AGR2 localizes to the lumen of the ER and indirectly associates with ER membrane-bound ribosomes through nascent protein chains. We showed that AGR2 expression is controlled by the unfolded protein response and is in turn is involved in the maintenance of ER homeostasis. Remarkably, we have demonstrated that siRNA-mediated knockdown of AGR2 significantly alters the expression of components of the ER-associated degradation machinery and reduces the ability of cells to cope with acute ER stress, properties that might be relevant to the role of AGR2 in cancer development.

Pancreatic β-cells have a well-developed endoplasmic reticulum due to their highly specialized secretory function to produce insulin in response to glucose and nutrients. It has been previously reported that overexpression of activating transcription factor 6 (ATF6) reduces insulin gene expression in part via upregulation of small heterodimer partner. In this study, we investigated whether ATF6 directly binds to the insulin gene promoter, and whether its direct binding represses insulin gene promoter activity. A bioinformatics analysis identified a putative ATF6 binding site in the A5/Core region of the rat insulin II gene promoter. Direct binding of ATF6 was confirmed using several approaches. Electrophoretic mobility shift assays in nuclear extracts from MCF7 cells, isolated rat islets and insulin-secreting HIT-T15 cells showed ATF6 binding to the native A5/Core of the rat insulin II gene promoter. Antibody-mediated supershift analyses revealed the presence of both ATF6 isoforms, ATF6α and ATF6β, in the complex. Chromatin immunoprecipitation assays confirmed the binding of ATF6α and ATF6β to a region encompassing the A5/Core of the rat insulin II gene promoter in isolated rat islets. Overexpression of the active (cleaved) fragment of ATF6α, but not ATF6β, inhibited the activity of an insulin promoter-reporter by 50%. However, the inhibitory effect of ATF6α was insensitive to mutational inactivation or deletion of the A5/Core. Therefore, although ATF6 binds directly to the A5/Core of the rat insulin II gene promoter, this direct binding does not appear to contribute to its repressive activity.

© 2011 Society for Endocrinology

It is well known that the endoplasmic reticulum (ER) is capable of expanding its surface area in response both to cargo load and to increased expression of resident membrane proteins. Although the response to increased cargo load, known as the unfolded protein response (UPR), is well characterized, the mechanism of the response to membrane protein load has been unclear. As a model system to investigate this phenomenon, we have used a HeLa-TetOff cell line inducibly expressing a tail-anchored construct consisting of an N-terminal cytosolic GFP moiety anchored to the ER membrane by the tail of cytochrome b5 [GFP-b(5)tail]. After removal of doxycycline, GFP-b(5)tail is expressed at moderate levels (1-2% of total ER protein) that, nevertheless, induce ER proliferation, as assessed both by EM and by a three- to fourfold increase in phosphatidylcholine synthesis. We investigated possible participation of each of the three arms of the UPR and found that only the activating transcription factor 6 (ATF6) arm was selectively activated after induction of GFP-b(5)tail expression; peak ATF6α activation preceded the increase in phosphatidylcholine synthesis. Surprisingly, up-regulation of known ATF6 target genes was not observed under these conditions. Silencing of ATF6α abolished the ER proliferation response, whereas knockdown of Ire1 was without effect. Because GFP-b(5)tail lacks a luminal domain, the response we observe is unlikely to originate from the ER lumen. Instead, we propose that a sensing mechanism operates within the lipid bilayer to trigger the selective activation of ATF6.

PURPOSE:

Many rhodopsin mutations that cause retinitis pigmentosa produce misfolded rhodopsin proteins that are retained within the endoplasmic reticulum (ER) and cause photoreceptor cell death. Activating transcription factor 6 (ATF6) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) control intracellular signaling pathways that maintain ER homeostasis. The aim of this study was to investigate how ATF6 and PERK signaling affected misfolded rhodopsin in cells, which could identify new molecular therapies to treat retinal diseases associated with ER protein misfolding.

METHODS:

To examine the effect of ATF6 on rhodopsin, wild-type (WT) or mutant rhodopsins were expressed in cells expressing inducible human ATF6f, the transcriptional activator domain of ATF6. Induction of ATF6f synthesis rapidly activated downstream genes. To examine PERK's effect on rhodopsin, WT or mutant rhodopsins were expressed in cells expressing a genetically altered PERK protein, Fv2E-PERK. Addition of the dimerizing molecule (AP20187) rapidly activated Fv2E-PERK and downstream genes. By use of these strategies, it was examined how selective ATF6 or PERK signaling affected the fate of WT and mutant rhodopsins.

RESULTS:

ATF6 significantly reduced T17M, P23H, Y178C, C185R, D190G, K296E, and S334ter rhodopsin protein levels in the cells with minimal effects on monomeric WT rhodopsin protein levels. By contrast, the PERK pathway reduced both levels of WT, mutant rhodopsins, and many other proteins in the cell.

CONCLUSIONS:

This study indicates that selectively activating ATF6 or PERK prevents mutant rhodopsin from accumulating in cells. ATF6 signaling may be especially useful in treating retinal degenerative diseases arising from rhodopsin misfolding by preferentially clearing mutant rhodopsin and abnormal rhodopsin aggregates.

The unfolded protein response (UPR) is an adaptive stress response that responds to the accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER) and that adjusts the protein-folding capacity to the needs of the cell. Perturbation of cellular lipids also activates the UPR. Lipid-induced UPR has attracted much attention because it is associated with the pathology of some metabolic diseases. However, how the lipid-induced UPR is activated remains unclear. We previously showed that palmitic acid treatment or knockdown of stearoyl-CoA desaturase in HeLa cells promotes membrane lipid saturation and activates the UPR. In this study, we compared UPR activation by membrane lipid saturation with UPR activation by conventional ER stressors that cause the accumulation of unfolded proteins such as tunicamycin and thapsigargin. Membrane lipid saturation induced autophosphorylation of inositol-requiring 1α (IRE1α) and protein kinase RNA-like ER kinase, but not the conversion of activating transcription factor-6α to the active form. A conventional ER stressor induced clustering of fluorescently tagged IRE1α fusion protein, but palmitic acid treatment did not, suggesting that IRE1α was activated without large cluster formation by membrane lipid saturation. Together, these results suggest membrane lipid saturation, and unfolded proteins activate the UPR through different mechanisms.

© 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

The molecular mechanisms and cellular targets of sorafenib, a multikinase inhibitor used for the treatment of hepatocellular carcinoma (HCC), remain to be fully characterized. Recent studies have shown that sorafenib induces tumor cell death through the activation of endoplasmic reticulum stress signaling and/or autophagy in various cellular models. Using liver cancer-derived cell lines, we specifically show that the IRE1 and phosphorylated extracellular signal-regulated kinase arms of the unfolded protein response (UPR) become activated upon sorafenib treatment, whereas the ATF6 arm is inhibited. Our results also reveal that sorafenib treatment causes disruption to the secretory pathway, as witnessed by the fragmentation of the Golgi apparatus and the induction of autophagy. On the basis of these observations, we tested the relevance of the AAA⁺ ATPase p97/VCP as a potential functional target of sorafenib. Our results show that p97/VCP tyrosine phosphorylation is prevented upon sorafenib treatment, and that this can be correlated with enhanced membrane association. Moreover, we show that DBeQ, a recently discovered inhibitor of p97/VCP, enhances sorafenib-mediated toxicity in cultured cells. Our data show a novel mechanism for sorafenib-mediated cell death in HCC, which depends on the integrity of the secretory pathway; and we identify p97/VCP phosphorylation as a potential target for improved sorafenib treatment efficacy in patients.

The endoplasmic reticulum (ER), first compartment of the secretory pathway, is mainly involved in calcium sequestration and lipid biosynthesis and in the translation, folding, and transport of secretory proteins. Under some physiological and physiopathological situations, secretory proteins do not acquire their folded conformation and accumulate in the ER. An adaptive response named the UPR is then triggered from this compartment to restore its homeostasis. In the past few years, interconnections between the UPR and small GTPase signaling have been established. In an attempt to further investigate these novel signaling networks, we hereby provide a detailed description of experimental strategies available. We describe in detail methods to monitor both UPR and small GTPase signaling and the outcomes of such approaches in the identification of new links between those signaling pathways using pharmacological and genetic screens. In physiopathological contexts, the guidelines herein should enable researchers in the field to establish essential means for determination of functional interactions between those pathways.

Copyright © 2011 Elsevier Inc. All rights reserved.