Quantitative interactome proteomics identifies a proteostasis network for GABAA receptors

Wang, Yajuan; Di, Xiao-Jing; Mu, Ting-Wei

doi:10.1016/j.jbc.2022.102423

Cited by 11 publications

(17 citation statements)

References 88 publications

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“…While these networks remain largely unknown for K V 11.1, research has illuminated the networks for several other ion channel disorders. 14,15,30,31…”

Section: Resultsmentioning

confidence: 99%

“…Several mitochondrial proteins were found to interact with gamma-aminobutyric acid type A (GABA A ) channels as well and warrant further exploration into possible functional roles on K V 11.1 biogenesis. 31 The interaction with HSD17B12, which converts estrone to estradiol could be particularly significant given that female sex hormones increase the risk of LQTS related arrhythmias. 44,45 Several other 17-beta hydroxysteroid dehydrogenases localize in the ER membrane, and the catalytic site of HSD17B12 was found facing the ER lumen.…”

Section: Discussionmentioning

confidence: 99%

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The proteostasis interactomes of trafficking-deficient K_V11.1 variants associated with Long QT Syndrome and pharmacological chaperone rescue

Egly,

Barny,

et al. 2024

Preprint

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Introduction: 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. Methods: We used affinity-purification coupled with tandem mass tag-based quantitative mass spectrometry to assess protein interaction changes in human embryonic kidney (HEK293) cells expressing wild-type (WT) KV11.1 or trafficking-deficient channel variants in the presence or absence of E 4031. Results: We identified 573 core KV11.1 protein interactors. Both variants KV11.1-G601S and KV11.1-G601S-G965* had significantly increased interactions with proteins responsible for folding, trafficking, and degradation compared to WT. We found that proteasomal degradation is a key component for KV11.1 degradation and that the KV11.1-G601S-G965* variant was more responsive to E-4031 treatment. This suggests a role in the C-terminal domain and the ER retention motif of KV11.1 in regulating trafficking. Conclusion: Our report characterizes the proteostasis network of KV11.1, two trafficking deficient KV11.1 variants, and variants treated with a pharmacological chaperone. The identified protein interactions could be targeted therapeutically to improve KV11.1 trafficking and treat Long QT Syndrome.

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“…While these networks remain largely unknown for K V 11.1, research has illuminated the networks for several other ion channel disorders. 14,15,30,31…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

The proteostasis interactomes of trafficking-deficient K_V11.1 variants associated with Long QT Syndrome and pharmacological chaperone rescue

Egly,

Barny,

et al. 2024

Preprint

View full text Add to dashboard Cite

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“…PC treatment adapts the ER proteostasis by adjusting the interactions between DAVs and GABA A receptor proteostasis network (Fig. 5-7) 13 : the interactions between the variants and pro-folding chaperones (BiP and calnexin) and a trafficking factor (LMAN1) are enhanced, whereas the interactions between the variants and ERAD factors (Grp94 and VCP) are reduced by PC chaperone treatment (Fig 8). Since Hispidulin and TP003 are PAMs of GABA A receptors, in addition to their PC effects on restoring the trafficking of GABA A variants, presumably, their application could potentially increase GABA-induced currents further once the receptors are able to reach the plasma membrane, adding the benefits of using these compounds to correct the function of GABA A variants.…”

Section: Discussionmentioning

confidence: 99%

“…Since the α1 subunit has two glycosylation sites (Asn38 and Asn138) in the ER, endo H digestion produced two endo Hresistant bands (Fig. 5c Since the ER proteostasis network orchestrates the folding, assembly, degradation, and trafficking of GABA A receptors 13 , we determined the effect of Hispidulin or TP003 on adapting this network. Previously, we demonstrated that ER resident chaperones, including BiP and calnexin, positively regulate the productive folding of GABA A receptors 15,16 , and LMAN1 (aka ERGIC53) promotes their anterograde trafficking 18 .…”

Section: Hispidulin and Tp003 Promote Functional Surface Expression O...mentioning

confidence: 99%

“…The proteostasis network orchestrates the folding, assembly, trafficking, and degradation of GABA A receptors. Recently, our quantitative proteomics identified a proteostasis network for GABA A receptors, which enabled our efforts to further elucidate their biogenesis pathways 13 . Upon the synthesis of GABA A receptor subunits, the ER membrane protein complex (EMC), including EMC3 and EMC6, facilitates the insertion of the transmembrane domain of GABA A receptors into the ER membrane 14 .…”

Section: Introductionmentioning

confidence: 99%

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Pharmacological chaperones restore proteostasis of epilepsy-associated GABA_Areceptor variants

Wang

Seibert

Ahn

et al. 2023

Preprint

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Recent advances in genetic diagnosis identified variants in genes encoding GABAA receptors as causative for genetic epilepsy. Here, we selected eight disease-associated variants in the alpha1 subunit of GABAA receptors causing mild to severe clinical phenotypes and showed that they are loss of function, mainly by reducing the folding and surface trafficking of the alpha1 protein. Furthermore, we sought client protein-specific pharmacological chaperones to restore the function of pathogenic receptors. Applications of positive allosteric modulators, including Hispidulin and TP003, increase the functional surface expression of the alpha1 variants. Mechanism of action study demonstrated that they enhance the folding and assembly and reduce the degradation of GABAA variants without activating the unfolded protein response in HEK293T cells and human iPSC-derived neurons. Since these compounds cross the blood-brain barrier, such a pharmacological chaperoning strategy holds great promise to treat genetic epilepsy in a GABAA receptor-specific manner.

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Pathogenicity Prediction of GABA_A Receptor Missense Variants

Wang,

Vu,

2024

Israel Journal of Chemistry

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Variants in the genes encoding gamma‐aminobutyric acid type A (GABAA) receptor subunits are associated with epilepsy. To date, over 1000 clinical variants have been identified in these genes. However, the majority of these variants lack functional studies and their clinical significance is uncertain although accumulating evidence indicates that proteostasis deficiency is the major disease‐causing mechanism. Here, we apply two state‐of‐the‐art modeling tools, namely AlphaMissense and Rhapsody to predict the pathogenicity of saturating missense variants in genes that encode the major subunits of GABAA receptors in the central nervous system, including GABRA1, GABRB2, GABRB3, and GABRG2. We demonstrate that the predicted pathogenicity correlates well between AlphaMissense and Rhapsody. In addition, AlphaMissense pathogenicity score correlates modestly with plasma membrane expression, peak current amplitude, and GABA potency of the variants that have available experimental data. Furthermore, almost all annotated pathogenic variants in the ClinVar database are successfully identified from the prediction, whereas uncertain variants from ClinVar partially due to the lack of experimental data are differentiated into different pathogenicity groups. The pathogenicity prediction of GABAA receptor missense variants provides a resource to the community as well as guidance for future experimental and clinical investigations.

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Quantitative interactome proteomics identifies a proteostasis network for GABAA receptors

Cited by 11 publications

References 88 publications

The proteostasis interactomes of trafficking-deficient K_V11.1 variants associated with Long QT Syndrome and pharmacological chaperone rescue

The proteostasis interactomes of trafficking-deficient K_V11.1 variants associated with Long QT Syndrome and pharmacological chaperone rescue

Pharmacological chaperones restore proteostasis of epilepsy-associated GABA_Areceptor variants

Pathogenicity Prediction of GABA_A Receptor Missense Variants

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Quantitative interactome proteomics identifies a proteostasis network for GABAA receptors

Cited by 11 publications

References 88 publications

The proteostasis interactomes of trafficking-deficient KV11.1 variants associated with Long QT Syndrome and pharmacological chaperone rescue

The proteostasis interactomes of trafficking-deficient KV11.1 variants associated with Long QT Syndrome and pharmacological chaperone rescue

Pharmacological chaperones restore proteostasis of epilepsy-associated GABAAreceptor variants

Pathogenicity Prediction of GABAA Receptor Missense Variants

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Product

Resources

About

The proteostasis interactomes of trafficking-deficient K_V11.1 variants associated with Long QT Syndrome and pharmacological chaperone rescue

The proteostasis interactomes of trafficking-deficient K_V11.1 variants associated with Long QT Syndrome and pharmacological chaperone rescue

Pharmacological chaperones restore proteostasis of epilepsy-associated GABA_Areceptor variants

Pathogenicity Prediction of GABA_A Receptor Missense Variants