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The inducuction of UPR target genes in response to zinc depletion and serum starvation in HeLa cells.

ABSTRACT: Zinc is an essential trace element, and impaired zinc homeostasis is implicated in the pathogenesis of various human diseases. However, the mechanisms cells use to respond to zinc deficiency are poorly understood. We previously reported that amyotrophic lateral sclerosis (ALS)-linked pathogenic mutants of SOD1 cause chronic ER stress through specific interactions with Derlin-1, which is a component of the ER-associated degradation machinery. Moreover, we recently demonstrated that this interaction is common to ALS-linked SOD1 mutants, and SOD1WT comprises a masked Derlin-1-binding region. We found that under serum starved and zinc-deficient conditions, SOD1WT adopts a mutant-like conformation that exposes the Derlin-1-binding region, suggesting that SOD1-Derlin-1 interaction may contribute to induce ER stress under these conditions. Using RNA microarrays, we revealed that the zinc depletion and serum starvation induce the expression of the unfolded protein response (UPR) target genes, including HSPA5/BiP, SEL1L, DNAJC3/p58, and ARMET. We conclude that SOD1 has a novel function as a molecular switch that activates the ER stress response under zinc-deficient conditions

ORGANISM(S): Homo sapiens

PROVIDER: GSE49657 | GEO | 2013/08/09

SECONDARY ACCESSION(S): PRJNA214661

REPOSITORIES: GEO

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The inducuction of UPR target genes in response to zinc depletion and serum starvation in HeLa cells.

Project description:Zinc is an essential trace element, and impaired zinc homeostasis is implicated in the pathogenesis of various human diseases. However, the mechanisms cells use to respond to zinc deficiency are poorly understood. We previously reported that amyotrophic lateral sclerosis (ALS)-linked pathogenic mutants of SOD1 cause chronic ER stress through specific interactions with Derlin-1, which is a component of the ER-associated degradation machinery. Moreover, we recently demonstrated that this interaction is common to ALS-linked SOD1 mutants, and SOD1WT comprises a masked Derlin-1-binding region. We found that under serum starved and zinc-deficient conditions, SOD1WT adopts a mutant-like conformation that exposes the Derlin-1-binding region, suggesting that SOD1-Derlin-1 interaction may contribute to induce ER stress under these conditions. Using RNA microarrays, we revealed that the zinc depletion and serum starvation induce the expression of the unfolded protein response (UPR) target genes, including HSPA5/BiP, SEL1L, DNAJC3/p58, and ARMET. We conclude that SOD1 has a novel function as a molecular switch that activates the ER stress response under zinc-deficient conditions HeLa cells were treated with 8M-BM-5M TPEN for 9h or exposured to 36h of serum starvation with or without 30M-BM-5M zinc.Then the total RNA was isolated from cells using the Isogen reagent (Wako) following the manufacturer's recommendations.

2013-08-09 | E-GEOD-49657 | biostudies-arrayexpress

Sel1L is Indispensable for Mammalian ERAD, ER Homeostasis and Survival

Project description:Sel1L is an adaptor protein for the E3 ligase Hrd1 involved in endoplasmic reticulum-associated degradation (ERAD). Its physiological importance in mammalian ERAD, however, remains to be established. Here, using the inducible Sel1L knockout mouse and cell models, we provide the first in vivo evidence that Sel1L is indispensable for Hrd1 stability, ER homeostasis and survival. Acute loss of Sel1L leads to premature death in adult mice within 3 weeks with profound pancreatic atrophy. Contrary to current belief, our data show that mammalian Sel1L is required for Hrd1 stability and ERAD function both in vitro and in vivo. Sel1L deficiency disturbs ER homeostasis, activates ER stress, attenuates translation and promotes cell death. Serendipitously, using biochemical approach coupled with mass spectrometry, we found that Sel1L deficiency causes the aggregation of both small and large ribosomal subunits. Thus, Sel1L is an indispensable component of mammalian ERAD and ER homeostasis, which is essential for protein translation, pancreatic function, cellular and organismal survival. Pancreas tissue of wild type and inducible Sel1L knockout mice were subjected to gene expression analysis.

2013-12-04 | E-GEOD-52929 | biostudies-arrayexpress

IRE1alpha is an endogenous substrate of endoplasmic reticulum-associated degradation

Project description:Endoplasmic reticulum-associated degradation (ERAD) represents a principle quality control (QC) mechanism to clear misfolded proteins in the ER; however, its physiological significance and the nature of endogenous ERAD substrates remain largely unknown. Here we discover that IRE1alpha, the sensor of unfolded protein response (UPR), is a bona fide substrate of the Sel1L-Hrd1 ERAD complex. Mechanistically, ERAD-mediated IRE1alpha degradation occurs in a Bip-dependent manner under basal conditions and is attenuated in response to ER stress. Both intramembrane hydrophilic residues of IRE1alpha and lectin protein OS9 are required for IRE1alpha degradation. ERAD deficiency causes IRE1alpha protein stabilization, accumulation and mild activation both in vitro and in vivo, leading to cellular hypersensitivity to ER stress and inflammation. Furthermore, though enterocyte-specific Sel1L-knockout mice (Sel1LÎ?IEC) are viable and appear normal, they are more susceptible to experimental colitis in an IRE1alpha-dependent but CHOP-independent manner. Collectively, these results demonstrate that Sel1L-Hrd1 ERAD serves a distinct, essential function in restraint of IRE1alpha signaling in vivo by managing its protein turnover. Colon epithelium of wild type and enterocyte-specific Sel1L knockout mice were subjected to gene expression analysis.

2015-11-04 | E-GEOD-70563 | biostudies-arrayexpress

Sel1L is Indispensable for Mammalian ERAD, ER Homeostasis and Survival

2013-12-04 | GSE52929 | GEO

Hepatic Sel1L-Hrd1 ER-associated degradation deficiency leads to intrahepatic cholestasis in mice

Project description:The liver is the central organ critically regulating the balance of the metabolically potent yet toxic bile acids in the body. While genomic association studies have pointed to hepatic Sel1L – a critical component of mammalian Hrd1 ER-associated degradation (ERAD) machinery – as an influencer of serum bile acid levels, physiological relevance and mechanistic insights of ERAD in bile homeostasis remain unexplored. Using hepatocyte-specific Sel1L-deficient mouse models, we report that hepatic Sel1L-Hrd1 ERAD critically manages bile homeostasis in the body. Mice with hepatocyte-specific Sel1L developed intrahepatic cholestasis, with significant overload of bile acids in the liver and circulation under basal condition, and were hypersensitive to dietary bile acid challenge. By contrast, biliary bile acid and phosphatidylcholine levels were reduced, pointing to an export defect from hepatocytes. Unbiased proteomics analysis followed by biochemical assays revealed significant accumulation of the bile-stabilizing phosphatidylcholine exporter ATP-binding cassette 4 (Abcb4) in the ER of Sel1L-deficient livers, a gene associated with Progressive Familial Intrahepatic Cholestasis type III. Indeed, Abcb4 was a substrate of Sel1L-Hrd1 ERAD. Hence, hepatic Sel1L-Hrd1 ERAD maintains bile equilibrium via quality control of Abcb4 maturation in the ER.

2023-03-11 | PXD035243 | Pride

IRE1alpha is an endogenous substrate of endoplasmic reticulum-associated degradation

Project description:Endoplasmic reticulum-associated degradation (ERAD) represents a principle quality control (QC) mechanism to clear misfolded proteins in the ER; however, its physiological significance and the nature of endogenous ERAD substrates remain largely unknown. Here we discover that IRE1alpha, the sensor of unfolded protein response (UPR), is a bona fide substrate of the Sel1L-Hrd1 ERAD complex. Mechanistically, ERAD-mediated IRE1alpha degradation occurs in a Bip-dependent manner under basal conditions and is attenuated in response to ER stress. Both intramembrane hydrophilic residues of IRE1alpha and lectin protein OS9 are required for IRE1alpha degradation. ERAD deficiency causes IRE1alpha protein stabilization, accumulation and mild activation both in vitro and in vivo, leading to cellular hypersensitivity to ER stress and inflammation. Furthermore, though enterocyte-specific Sel1L-knockout mice (Sel1LΔIEC) are viable and appear normal, they are more susceptible to experimental colitis in an IRE1alpha-dependent but CHOP-independent manner. Collectively, these results demonstrate that Sel1L-Hrd1 ERAD serves a distinct, essential function in restraint of IRE1alpha signaling in vivo by managing its protein turnover.

2015-11-04 | GSE70563 | GEO

Misfolded SOD1 binds the ubiquitin ligase TRAF6

Project description:Amyotrophic lateral sclerosis (ALS) is a fatal disease, characterized by the selective loss of motor neurons. Mutations in Cu/Zn superoxide dismutase (SOD1) are the second most common cause of ALS and it is now well accepted that they result in a gain of toxicity due to protein misfolding. We previously demonstrated in the SOD1G93A rat model that misfolded SOD1 exists as distinct conformers and deposits on mitochondrial subpopulations. Here, using SOD1G93A rats coupled with conformation-restricted antibodies for misfolded SOD1 (AMF7-63 and B8H10), we identified the interactomes of the mitochondrial pools of misfolded SOD1. This strategy identified binding partners that uniquely interacted with either AMF7-63- or B8H10-reactive SOD1 conformers as well as a high proportion of interactors common to both conformers. Of this latter set, we identified the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6) and determined that exposure of the SOD1 functional loops facilitates this interaction. However, this conformational criterion is not universally fulfilled by all SOD1 mutants and differentiates TRAF6-interacting from non-interacting SOD1 mutants. Functionally, TRAF6 stimulates mutant SOD1 polyubiquitination and aggregation. While TRAF6 E3 ubiquitin ligase activity is required for the former, it is dispensable for the latter, indicating that the polyubiquitination and aggregation of mutant SOD1 mediated by TRAF6 are independent events. We propose that misfolded SOD1 binding to TRAF6 may be relevant to ALS disease.

2020-02-24 | PXD015279 | Pride

Pathways Disrupted in Human ALS Motor Neurons Identified Through Genetic Correction of Mutant SOD1

Project description:Although many distinct mutations in a variety of genes are known to cause Amyotrophic Lateral Sclerosis (ALS), it remains poorly understood how they selectively impact motor neuron biology and whether they converge on common pathways to cause neural degeneration. Here, we have combined reprogramming and stem cell differentiation approaches with genome engineering and RNA sequencing to define the transcriptional changes that are induced in human motor neurons by mutant SOD1. Mutant SOD1 protein induced a transcriptional signature indicative of increased oxidative stress, reduced mitochondrial function, altered sub-cellular transport as well as activation of the ER stress and unfolded protein response pathways. Functional studies demonstrated that perturbations in these pathways were indeed the source of altered transcript levels.

2014-02-03 | GSE54409 | GEO

ER-associated degradation adapter Sel1L is required for CD8+ T cell function and memory formation following acute viral infection

Project description:The maintenance and persistence of antigen-specific CD8+ T cells underlies the efficacy of vaccines and immunotherapies. Pathways that contribute to the loss of CD8+ T cells are not completely understood. Uncovering the pathways underlying the limited persistence of CD8+ T cells would be of significant benefit for developing novel strategies of promoting T cell persistence and identifying patients whose T cells could function best for treatment. Here, we demonstrate the experience of endoplasmic reticulum (ER) stress in differentiating murine CD8+ T cells in vitro and in vivo. ER-associated degradation (ERAD) adapter Sel1L was found to be selectively induced in activated CD8+ T cells. Loss of Sel1L limited CD8+ T cell function and memory formation following acute viral infection. Mechanistically, Sel1L is required for optimal T cell oxidative metabolism and mitochondrial fusion. Finally, we demonstrate that human CD8+ T cells experience ER stress after activation and that ER stress is negatively associated with improved T cell functionality in bispecific antibody therapy and chimeric antigen receptor T cell therapy. Together these results demonstrated that ER stress and ERAD are important and novel regulators of T cell function and persistence.

2024-04-25 | GSE244315 | GEO

Amyotrophic Lateral Sclerosis-associated Mutants of SOD1 Modulate miRNA Biogenesis through Aberrant Interactions with Exportin 5

Project description:Mutations in SOD1 (Superoxide Dismutase 1) gene are associated with amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. By employing ascorbate peroxidase (APEX)-based proximity labeling, coupled with LC-MS/MS analysis, we uncovered 37 and 28 proteins exhibiting higher abundance in the proximity proteomes of SOD1G85R and SOD1G93A, respectively, than that of the wild-type SOD1. Immunoprecipitation followed by western blot analysis confirmed the preferential binding of one of these proteins, exportin 5 (XPO5), toward the two mutants of SOD1 over its wild-type counterpart. In line with the established function of XPO5 in pre-miRNA transport, we observed diminished nucleocytoplasmic transport of pre-miRNAs in cells with ectopic expression of the two SOD1 mutants over those expressing the wild-type counterpart. On the other hand, RT-qPCR results revealed significant elevations in mature miRNA in cells expressing the two SOD1 mutants, which are attributed to diminished inhibitory effect of XPO5 on Dicer-mediated cleavage of pre-miRNA to mature miRNA. Together, our chemoproteomic approach led to the revelation of a novel mechanism through which ALS-associated mutants of SOD1 perturb miRNA biogenesis, i.e., through aberrant binding toward XPO5.

2024-01-26 | PXD035452 | Pride

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