Misfolding, altered membrane distributions, and the unfolded protein response contribute to pathogenicity differences in Na,K-ATPase ATP1A3 mutations

Missense mutations in ATP1A3, the α3 isoform of Na,K-ATPase, cause neurological phenotypes that differ greatly in symptoms and severity. A mechanistic basis for differences is lacking, but reduction of activity alone cannot explain them. Isogenic cell lines with endogenous α1 and inducible exogenous α3 were constructed to compare mutation properties. Na,K-ATPase is made in endoplasmic reticulum, but glycan-free catalytic α subunit complexes with glycosylated β subunit in the ER to proceed through Golgi and post-Golgi trafficking. We previously observed classic evidence of protein misfolding in mutations with severe phenotypes: differences in ER retention of endogenous β1 subunit, impaired trafficking of α3, and cytopathology, suggesting that they misfold during biosynthesis. Here we tested two mutations associated with different phenotypes: D923N, which has a median age of onset of hypotonia or dystonia at 3 years, and L924P, with severe infantile epilepsy and profound impairment. Misfolding during biosynthesis in the ER activates the unfolded protein response (UPR), a multi-armed program that enhances protein folding capacity, and if that fails, triggers apoptosis. L924P showed more nascent protein retention in ER than D923N; more ER-associated degradation of α3 (ERAD); larger differences in Na,K-ATPase subunit distributions among subcellular fractions; and greater inactivation of eIF2α, a major defensive step of the UPR. In L924P there was also altered subcellular distribution of endogenous α1 subunit, analogous to a dominant negative effect. Both mutations showed pro-apoptotic sensitization by reduced phosphorylation of BAD. Encouragingly, however, 4-phenylbutyrate (4PBA), a pharmacological corrector, reduced L924P ER retention, increased α3 expression, and restored morphology.

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IRE1β negatively regulates IRE1α signaling in response to endoplasmic reticulum stress

The Journal of Cell Biology ◽

10.1083/jcb.201904048 ◽

2020 ◽

Vol 219 (2) ◽

Cited By ~ 4

Author(s):

Michael J. Grey ◽

Eva Cloots ◽

Mariska S. Simpson ◽

Nicole LeDuc ◽

Yevgeniy V. Serebrenik ◽

...

Keyword(s):

Epithelial Cells ◽

Er Stress ◽

Unfolded Protein Response ◽

Kinase Domain ◽

Dominant Negative ◽

Hek293 Cells ◽

Stress Sensor ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response

IRE1β is an ER stress sensor uniquely expressed in epithelial cells lining mucosal surfaces. Here, we show that intestinal epithelial cells expressing IRE1β have an attenuated unfolded protein response to ER stress. When modeled in HEK293 cells and with purified protein, IRE1β diminishes expression and inhibits signaling by the closely related stress sensor IRE1α. IRE1β can assemble with and inhibit IRE1α to suppress stress-induced XBP1 splicing, a key mediator of the unfolded protein response. In comparison to IRE1α, IRE1β has relatively weak XBP1 splicing activity, largely explained by a nonconserved amino acid in the kinase domain active site that impairs its phosphorylation and restricts oligomerization. This enables IRE1β to act as a dominant-negative suppressor of IRE1α and affect how barrier epithelial cells manage the response to stress at the host–environment interface.

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The Human Cytomegalovirus Endoplasmic Reticulum-Resident Glycoprotein UL148 Activates the Unfolded Protein Response

Journal of Virology ◽

10.1128/jvi.00896-18 ◽

2018 ◽

Vol 92 (20) ◽

Cited By ~ 13

Author(s):

Mohammed N. A. Siddiquey ◽

Hongbo Zhang ◽

Christopher C. Nguyen ◽

Anthony J. Domma ◽

Jeremy P. Kamil

Keyword(s):

Endoplasmic Reticulum ◽

Unfolded Protein Response ◽

Human Cytomegalovirus ◽

Mrna Translation ◽

Wild Type ◽

Α Subunit ◽

Unfolded Protein ◽

Eif2α Phosphorylation ◽

The Unfolded Protein Response ◽

Protein Response

ABSTRACTEukaryotic cells are equipped with three sensors that respond to the accumulation of misfolded proteins within the lumen of the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR), which functions to resolve proteotoxic stresses involving the secretory pathway. Here, we identify UL148, a viral ER-resident glycoprotein from human cytomegalovirus (HCMV), as an inducer of the UPR. Metabolic labeling results indicate that global mRNA translation is decreased when UL148 expression is induced in uninfected cells. Further, we find that ectopic expression of UL148 is sufficient to activate at least two UPR sensors: the inositol-requiring enzyme-1 (IRE1), as indicated by splicing ofXbp-1mRNA, and the protein kinase R (PKR)-like ER kinase (PERK), as indicated by phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) and accumulation of activating transcription factor 4 (ATF4). During wild-type HCMV infection, increases inXbp-1splicing, eIF2α phosphorylation, and accumulation of ATF4 accompany UL148 expression.UL148-null infections, however, show reduced levels of these UPR indicators and decreases in XBP1s abundance and in phosphorylation of PERK and IRE1. Small interfering RNA (siRNA) depletion of PERK dampened the extent of eIF2α phosphorylation and ATF4 induction observed during wild-type infection, implicating PERK as opposed to other eIF2α kinases. A virus withUL148disrupted showed significant 2- to 4-fold decreases during infection in the levels of transcripts canonically regulated by PERK/ATF4 and by the ATF6 pathway. Taken together, our results argue that UL148 is sufficient to activate the UPR when expressed ectopically and that UL148 is an important cause of UPR activation in the context of the HCMV-infected cell.IMPORTANCEThe unfolded protein response (UPR) is an ancient cellular response to ER stress that is of broad importance to viruses. Certain consequences of the UPR, including mRNA degradation and translational shutoff, would presumably be disadvantageous to viruses, while other attributes of the UPR, such as ER expansion and upregulation of protein folding chaperones, might enhance viral replication. Although HCMV is estimated to express well over 150 different viral proteins, we show that the HCMV ER-resident glycoprotein UL148 contributes substantially to the UPR during infection and, moreover, is sufficient to activate the UPR in noninfected cells. Experimental activation of the UPR in mammalian cells is difficult to achieve without the use of toxins. Therefore, UL148 may provide a new tool to investigate fundamental aspects of the UPR. Furthermore, our findings may have implications for understanding the mechanisms underlying the effects of UL148 on HCMV cell tropism and evasion of cell-mediated immunity.

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Feedback Inhibition of the Unfolded Protein Response by GADD34-Mediated Dephosphorylation of eIF2α

The Journal of Cell Biology ◽

10.1083/jcb.153.5.1011 ◽

2001 ◽

Vol 153 (5) ◽

pp. 1011-1022 ◽

Cited By ~ 854

Author(s):

Isabel Novoa ◽

Huiqing Zeng ◽

Heather P. Harding ◽

David Ron

Keyword(s):

Unfolded Protein Response ◽

Feedback Inhibition ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Α Subunit ◽

Unfolded Protein ◽

New Genes ◽

The Unfolded Protein Response ◽

Stress Dependent ◽

Protein Response

Phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) on serine 51 integrates general translation repression with activation of stress-inducible genes such as ATF4, CHOP, and BiP in the unfolded protein response. We sought to identify new genes active in this phospho-eIF2α–dependent signaling pathway by screening a library of recombinant retroviruses for clones that inhibit the expression of a CHOP::GFP reporter. A retrovirus encoding the COOH terminus of growth arrest and DNA damage gene (GADD)34, also known as MYD116 (Fornace, A.J., D.W. Neibert, M.C. Hollander, J.D. Luethy, M. Papathanasiou, J. Fragoli, and N.J. Holbrook. 1989. Mol. Cell. Biol. 9:4196–4203; Lord K.A., B. Hoffman-Lieberman, and D.A. Lieberman. 1990. Nucleic Acid Res. 18:2823), was isolated and found to attenuate CHOP (also known as GADD153) activation by both protein malfolding in the endoplasmic reticulum, and amino acid deprivation. Despite normal activity of the cognate stress-inducible eIF2α kinases PERK (also known as PEK) and GCN2, phospho-eIF2α levels were markedly diminished in GADD34-overexpressing cells. GADD34 formed a complex with the catalytic subunit of protein phosphatase 1 (PP1c) that specifically promoted the dephosphorylation of eIF2α in vitro. Mutations that interfered with the interaction with PP1c prevented the dephosphorylation of eIF2α and blocked attenuation of CHOP by GADD34. Expression of GADD34 is stress dependent, and was absent in PERK−/− and GCN2−/− cells. These findings implicate GADD34-mediated dephosphorylation of eIF2α in a negative feedback loop that inhibits stress-induced gene expression, and that might promote recovery from translational inhibition in the unfolded protein response.

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The Unfolded Protein Response in the Protozoan Parasite Toxoplasma gondii Features Translational and Transcriptional Control

Eukaryotic Cell ◽

10.1128/ec.00021-13 ◽

2013 ◽

Vol 12 (7) ◽

pp. 979-989 ◽

Cited By ~ 30

Author(s):

Bradley R. Joyce ◽

Zoi Tampaki ◽

Kami Kim ◽

Ronald C. Wek ◽

William J. Sullivan

Keyword(s):

Gene Expression ◽

Toxoplasma Gondii ◽

Er Stress ◽

Unfolded Protein Response ◽

Secretory Process ◽

Α Subunit ◽

Content Type ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response

ABSTRACT The unfolded protein response (UPR) is an important regulatory network that responds to perturbations in protein homeostasis in the endoplasmic reticulum (ER). In mammalian cells, the UPR features translational and transcriptional mechanisms of gene expression aimed at restoring proteostatic control. A central feature of the UPR is phosphorylation of the α subunit of eukaryotic initiation factor-2 (eIF2) by PERK (EIF2AK3/PEK), which reduces the influx of nascent proteins into the ER by lowering global protein synthesis, coincident with preferential translation of key transcription activators of genes that function to expand the processing capacity of this secretory organelle. Upon ER stress, the apicomplexan parasite Toxoplasma gondii is known to induce phosphorylation of Toxoplasma eIF2α and lower translation initiation. To characterize the nature of the ensuing UPR in this parasite, we carried out microarray analyses to measure the changes in the transcriptome and in translational control during ER stress. We determined that a collection of transcripts linked with the secretory process are induced in response to ER stress, supporting the idea that a transcriptional induction phase of the UPR occurs in Toxoplasma. Furthermore, we determined that about 500 gene transcripts showed enhanced association with translating ribosomes during ER stress. Many of these target genes are suggested to be involved in gene expression, including JmjC5, which continues to be actively translated during ER stress. This study indicates that Toxoplasma triggers a UPR during ER stress that features both translational and transcriptional regulatory mechanisms, which is likely to be important for parasite invasion and development.

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Dominant-Negative Calcium Channel Suppression by Truncated Constructs Involves a Kinase Implicated in the Unfolded Protein Response

Journal of Neuroscience ◽

10.1523/jneurosci.0553-04.2004 ◽

2004 ◽

Vol 24 (23) ◽

pp. 5400-5409 ◽

Cited By ~ 56

Author(s):

K. M. Page

Keyword(s):

Calcium Channel ◽

Unfolded Protein Response ◽

Dominant Negative ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response

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The Anti-Apoptotic Role of the Unfolded Protein Response in Bcr-Abl Positive Leukemia Cells.

Blood ◽

10.1182/blood.v110.11.4527.4527 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4527-4527

Author(s):

Atsuko Tanimura ◽

Toshiaki Yujiri ◽

Yoshinori Tanaka ◽

Yukio Tanizawa

Keyword(s):

Unfolded Protein Response ◽

Dominant Negative ◽

Leukemic Cells ◽

Luciferase Assay ◽

Leukemia Cells ◽

Unfolded Protein ◽

Ph Chromosome ◽

The Unfolded Protein Response ◽

Protein Response

Abstract Accumulation of unfolded or misfolded proteins within the endoplasmic reticulum triggers the unfolded protein response (UPR). Evidence from several studies suggests that the UPR is activated in various tumors and might play a crucial role in tumor growth. However, the role of the UPR in leukemia remains unclear. Therefore, to define the role of the UPR in leukemogenesis, we used p210 Bcr-Abl-expressing 32D myeloid cell lines (p210 32D). The mRNA expression of UPR-related genes, namely, a spliced form of X-box DNA-binding protein (XBP1) and glucose regulated protein 78 (GRP78), was increased in p210 32D. Luciferase assay indicated that p210 Bcr-Abl induced high levels of the transcriptional activity of the UPR element. Moreover, levels of the phosphorylated eIF2 alpha protein increased in p210 32D. Inhibition of the UPR using IRE1 and ATF6 dominant-negative mutants diminished the ability of Bcr-Abl to protect the cells from etoposide- and imatinib-induced apoptosis, but had no effect on the proliferation of p210 Bcr-Abl-transformed cells. We also evaluated the expression of UPR-related genes in primary leukemic cells from Ph chromosome-positive cells by real-time RT-PCR; it was found that these cells showed higher expression levels than the control cells. Taken together, these results for the first time suggested that UPR is a downstream target of the Bcr-Abl oncoprotein, and it plays the anti-apoptotic role of Bcr-Abl in Ph chromosome-positive leukemia cells.

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