Dataset Information

T he proteostasis interactomes of trafficking - deficient K V 11.1 variants associated with Long QT Syndrome and pharmacological chaperone rescue

ABSTRACT: The voltage gated potassium ion channel KV11.1 plays a critical role in cardiac repolarization. Genetic variants that render Kv11.1 dysfunctional cause Long QT Syndrome (LQTS), which is associated with fatal arrhythmias. Approximately 90% of LQTS-associated variants cause intracellular protein transport (trafficking) dysfunction, which can be rescued by pharmacological chaperones like E-4031. Protein folding and trafficking decisions are regulated by chaperones, protein quality control factors, and trafficking machinery comprising the cellular proteostasis network. Here, we test whether trafficking dysfunction is associated with alterations in the proteostasis network of pathogenic Kv11.1 variants, and whether pharmacological chaperones can normalize the proteostasis network of responsive variants.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Early Embryonic Cell, Kidney

DISEASE(S): Cardiovascular System Disease

SUBMITTER: Christian Egly

LAB HEAD: Lars Plate

PROVIDER: PXD050914 | Pride | 2024-08-10

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	12Sept2023_CE_Exp4_16plex_0C_1.raw	Raw
	12Sept2023_CE_Exp4_16plex_100C_7.raw	Raw
	12Sept2023_CE_Exp4_16plex_10B_8.raw	Raw
	12Sept2023_CE_Exp4_16plex_10C_2.raw	Raw
	12Sept2023_CE_Exp4_16plex_20C_3.raw	Raw

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Publications

The proteostasis interactomes of trafficking-deficient variants of the voltage-gated potassium channel K<sub>V</sub>11.1 associated with long QT syndrome.

Egly Christian L CL Barny Lea A LA Do Tri T McDonald Eli F EF Knollmann Björn C BC Plate Lars L

The Journal of biological chemistry 20240612 7

The voltage-gated potassium ion channel K<sub>V</sub>11.1 plays a critical role in cardiac repolarization. Genetic variants that render Kv11.1 dysfunctional cause long QT syndrome (LQTS), which is associated with fatal arrhythmias. Approximately 90% of LQTS-associated variants cause intracellular protein transport (trafficking) dysfunction, which pharmacological chaperones like E-4031 can rescue. Protein folding and trafficking decisions are regulated by chaperones, protein quality control facto ...[more]

PMID: 38876300

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Project description: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 selectively restoring aberrant ER proteostasis of pathologic, destabilized proteins through arm-selective UPR-activation. The unfolded protein response adapts endoplasmic reticulum (ER) proteostasis via stress-responsive transcription factors including XBP1s and ATF6. Here, R. Luke Wiseman and colleagues implement technology for the orthogonal, ligand-dependent activation of XBP1s and/or ATF6 in a single cell. They characterize how XBP1s and/or ATF6 activation impacts ER proteostasis pathway composition and function. Adapted ER environments influence the proteostasis of destabilized protein variants without affecting the endogenous proteome. The work informs the development of proteostasis environment-adapting therapeutics for protein misfolding-related diseases. In order to activate both XBP1s and ATF6 in the same cell, we incorporated DHFR.ATF6 and tet-inducible XBP1s into a HEK293T-REx cell line stably expressing the tet-repressor. The HEK293DYG control cell line expresses tet-inducible eGFP and DHFR.YFP and is used as a control to demonstrate that the addition of doxycycline (dox) and trimethoprim (TMP) do not induce UPR genes. HEK293DYG cells were treated for 12 h with vehicle or 1 μg/mL dox and 10 μM TMP in biological triplicate. Cells were harvested and RNA was extracted using the RNeasy Mini Kit (Qiagen). Genomic DNA was removed by on-column digestion using the RNase-free DNase Set (Qiagen). Data from HEK293DYG cells showed no significant overlap in the ligand-treated transcriptomes obtained from HEK293DAX cells.

Project description: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 selectively restoring aberrant ER proteostasis of pathologic, destabilized proteins through arm-selective UPR-activation. The unfolded protein response adapts endoplasmic reticulum (ER) proteostasis via stress-responsive transcription factors including XBP1s and ATF6. Here, R. Luke Wiseman and colleagues implement technology for the orthogonal, ligand-dependent activation of XBP1s and/or ATF6 in a single cell. They characterize how XBP1s and/or ATF6 activation impacts ER proteostasis pathway composition and function. Adapted ER environments influence the proteostasis of destabilized protein variants without affecting the endogenous proteome. The work informs the development of proteostasis environment-adapting therapeutics for protein misfolding-related diseases. In order to activate both XBP1s and ATF6 in the same cell, we incorporated DHFR.ATF6 and tet-inducible XBP1s into a HEK293T-REx cell line stably expressing the tet-repressor. Selection of a single colony resulted in the HEK293DAX cell line in which XBP1s is induced by doxycycline and DHFR.ATF6 is activated by trimethoprim (TMP; TMP-dependent DHFR.ATF6 activation in HEK293DAX cells will henceforth be referred to as ATF6 activation for simplicity). HEK293DAX cells were treated for 12 h with vehicle, 1 ?g/mL dox, 10 ?M TMP, or both in biological triplicate. Cells were harvested and RNA was extracted using the RNeasy Mini Kit (Qiagen). Genomic DNA was removed by on-column digestion using the RNase-free DNase Set (Qiagen). Data from HEK293DYG cells showed no significant overlap in the ligand-treated transcriptomes obtained from the control HEK293DYG cells.

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Dataset Information

T he proteostasis interactomes of trafficking - deficient K V 11.1 variants associated with Long QT Syndrome and pharmacological chaperone rescue

Dataset's files

Publications

The proteostasis interactomes of trafficking-deficient variants of the voltage-gated potassium channel K<sub>V</sub>11.1 associated with long QT syndrome.

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Dataset Information

T he proteostasis interactomes of trafficking - deficient K V 11.1 variants associated with Long QT Syndrome and pharmacological chaperone rescue

Dataset's files

Publications

The proteostasis interactomes of trafficking-deficient variants of the voltage-gated potassium channel K&lt;sub&gt;V&lt;/sub&gt;11.1 associated with long QT syndrome.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

The proteostasis interactomes of trafficking-deficient variants of the voltage-gated potassium channel K<sub>V</sub>11.1 associated with long QT syndrome.