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Transient expression of misfolded surfactant protein C

ABSTRACT: Mutations in the SFTPC gene associated with interstitial lung disease in human patients result in misfolding, endoplasmic reticulum (ER) retention, and degradation of the encoded surfactant protein C (SP-C) proprotein. To identify candidate genes involved in ER quality control of SP-C, HEK293 cells were transiently transfected with mutant SP-C (SP-CΔexon4 or SP-CL188Q), SP-CWT, or vector cDNAs, and global changes in gene expression were assessed by microarray analyses. Microarray analysis demonstrated that the SPC exon 4 deletion and SPC L188Q mutations invoke very similar transcriptional profiles including the activation of major players mediating ER response and unfolding protein response (UPR) in transient transfection system. In combination with promoter scan (UPRE, ERSE, XBP1 sites) and protein domain analysis (Finding ER Lumen, ER Membrane retention signal, J-Domain and Leucine Zipper domain), we were able to not only verify the known ERAD components (XBP1, Bip, Erdj4&5), but also identify multiple ER components which may play critical roles in the detection and /or degradation of mutant SPC, which in turn will help us to gain better understanding of the entire mammalian ERAD machinery.

ORGANISM(S): Homo sapiens

PROVIDER: GSE16054 | GEO | 2009/05/14

SECONDARY ACCESSION(S): PRJNA114999

REPOSITORIES: GEO

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Transient expression of misfolded surfactant protein C

Project description:Mutations in the SFTPC gene associated with interstitial lung disease in human patients result in misfolding, endoplasmic reticulum (ER) retention, and degradation of the encoded surfactant protein C (SP-C) proprotein. To identify candidate genes involved in ER quality control of SP-C, HEK293 cells were transiently transfected with mutant SP-C (SP-CΔexon4 or SP-CL188Q), SP-CWT, or vector cDNAs, and global changes in gene expression were assessed by microarray analyses. Microarray analysis demonstrated that the SPC exon 4 deletion and SPC L188Q mutations invoke very similar transcriptional profiles including the activation of major players mediating ER response and unfolding protein response (UPR) in transient transfection system. In combination with promoter scan (UPRE, ERSE, XBP1 sites) and protein domain analysis (Finding ER Lumen, ER Membrane retention signal, J-Domain and Leucine Zipper domain), we were able to not only verify the known ERAD components (XBP1, Bip, Erdj4&5), but also identify multiple ER components which may play critical roles in the detection and /or degradation of mutant SPC, which in turn will help us to gain better understanding of the entire mammalian ERAD machinery. samples consisted of HEK293 cells transiently transfected with wild-type SP-C (WT), the delta exon4 mutant of SP-C (Ex4) or L188Q mutant of SP-C (LQ). Groups were performed in triplicate.

2009-05-14 | E-GEOD-16054 | biostudies-arrayexpress

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Project description:Mutations in the gene encoding surfactant protein C (SFTPC) have been linked to interstitial lung disease in children and adults. Expression of the index mutation, SP-Cdeltaexon4, in transiently transfected cells and type II cells of transgenic mice resulted in misfolding of the proprotein, activation of ER stress pathways and cytotoxicity. In the current study we show that stably transfected cells adapted to chronic ER stress imposed by constitutive expression of SP-Cdeltaexon4 via an NF-kB-dependent pathway. However, infection of cells expressing SP-Cdeltaexon4 with respiratory syncytial virus resulted in significantly enhanced cytotoxicity associated with accumulation of the mutant proprotein, pronounced activation of the unfolded protein response and cell death. Adaptation to chronic ER stress imposed by misfolded SP-C was associated with increased susceptibility to viral-induced cell death. The wide variability in the age of onset of ILD in patients with SFTPC mutations may be related to exposure to an environmental insult that ultimately overwhelms the homeostatic, cytoprotective response. Keywords: transcriptional response to misfolded protein due to genetic modification

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IRE1alpha is an endogenous substrate of endoplasmic reticulum-associated degradation

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2021-07-05 | PXD022002 | Pride

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.

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Sel1L is Indispensable for Mammalian ERAD, ER Homeostasis and Survival

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