Biology subjects

Mu, T.

Publications and source records attributed to Mu, T..

9 recordsLinked to original sources

bioRxiv2025

Phenylhydrazone-based Endoplasmic Reticulum Proteostasis Regulator Compounds with Enhanced Biological Activity

Pharmacological enhancement of endoplasmic reticulum (ER) proteostasis is an attractive strategy to mitigate pathology linked to etiologically-diverse protein misfolding diseases. However, despite this promise, few compounds have been identified that enhance ER proteostasis through defined mechanisms of action. We previously identified the phenylhydrazone-based compound AA263 as a compound that promotes adaptive ER proteostasis remodeling through mechanisms including preferential activation of the ATF6 signaling arm of the unfolded protein response (UPR). However, the protein target(s) of AA263 and the potential for further development of this class of ER proteostasis regulators had not been previously explored. Here, we employ chemical proteomics to demonstrate that AA263 covalently targets a subset of ER protein disulfide isomerases, revealing a potential molecular mechanism for the activation of ATF6 afforded by this compound. We then use medicinal chemistry to establish next-generation AA263 analogs showing improved potency and efficacy for ATF6 activation, as compared to the parent compound. Finally, we show that treatment with these AA263 analogs enhances secretory pathway proteostasis to correct the pathologic protein misfolding and trafficking of both a destabilized, disease-associated 1-antitrypsin (A1AT) variant and an epilepsy-associated GABAA receptor variant. These results establish AA263 analogs with enhanced potential for correcting imbalanced ER proteostasis associated with etiologically-diverse protein misfolding disorders.

cell biology↗

bioRxiv2025

Missense variants in GABAA receptor beta2 subunit disrupt receptor biogenesis and cause loss of function

Gamma-aminobutyric acid type A receptors (GABAARs) are the major inhibitory neurotransmitter-gated channel in the mammalian central nervous system. GABAARs function as heteropentamers, typically composed of two 1, two {beta}2, and one {gamma}2 subunits. Protein homeostasis between GABAAR folding, trafficking, assembly, and degradation is critical to ensure normal physiological functions. Variants in genes encoded for GABAARs lead to numerous neurological disorders, such as genetic epilepsy with or without neurodevelopmental delay. While these variants are associated with severe clinical presentations of epilepsy, the molecular mechanisms driving the disease remain to be elucidated. In this study, we focused on four missense epilepsy-associated variants (EAVs) in the GABRB2 gene: Q209F210delinsH (c. 627_629del), R240T (c. 719G>C), I246T (c. 737T>C), and I299S (c. 896T>G). HEK293T cells exogenously expressing these {beta}2 variants exhibited significantly reduced GABA-induced peak chloride current, indicating their loss of function. However, the four {beta}2 EAVs differed in the degree of proteostasis deficiencies, including increased ER retention, compromised assembly, decreased protein stability, and reduced trafficking and surface expression, with Q209F210delinsH and R240T variants leading to the most severe degradation. Collectively, these results indicate that these epilepsy-linked variants have debilitating effects on the early biogenesis of the {beta}2 subunit, causing misfolding, aggregation, and rapid degradation before it can be assembled with other subunits and transported to the plasma membrane. Overall, our work offers crucial mechanistic insight into how specific {beta}2 missense variants impact the proteostasis maintenance of GABAARs, which could facilitate the development of effective therapeutics for genetic epilepsy by targeting trafficking-deficient GABAAR variants.

cell biology↗

bioRxiv2025

A GluN2B disease-associated variant promotes degradation of NMDA receptors via autophagy

N-methyl-D-aspartate receptors (NMDARs) are essential for excitatory neurotransmission and their pathogenic variants can lead to proteostasis defects and thus neurological diseases. However, how the proteostasis network degrades pathogenic variants is not well understood. Here, we demonstrated that the R519Q GluN2B variant is retained in the endoplasmic reticulum (ER) and fails to traffic to the surface to form functional NMDARs. Pharmacological and genetic inhibition of autophagy results in the accumulation of this variant, indicating that it is degraded by the autophagy-lysosomal proteolysis pathway. Since GluN2B has a cytosolic LIR motif, which can interact with cytosolic autophagy machinery, we demonstrated that disrupting this LIR motif impairs the autophagic clearance of this variant. Additionally, the R519Q variant is recognized by ER-phagy receptors, including CCPG1 and RTN3L. Our result provides the molecular mechanism for the degradation of NMDAR variants and identifies a pathway for targeted therapeutic intervention for neurological disorders with dysfunctional NMDARs. SummaryNMDA receptors are essential for excitatory neurotransmission and their proteostasis defects lead to neurological diseases. Benske et al. report that pathogenic R519Q variants predispose GluN2B subunits to degradation and clearance by the autophagy-lysosomal pathway.

cell biology↗

bioRxiv2024

GABRA1 frameshift variants impair GABAA receptor proteostasis

The gamma-aminobutyric acid type A receptor (GABAAR) is the most common inhibitory neurotransmitter-gated ion channel in the central nervous system. Pathogenic variants in genes encoding GABAAR subunits can cause receptor dysfunction and lead to genetic epilepsy. Frameshift variants in these genes can result in a premature termination codon, producing truncated receptor subunit variants. However, the pathogenic molecular mechanism as well as functional implications of these frameshift variants remains inadequately characterized. This study focused on four clinical frameshift variants of the 1 subunit of GABAAR (encoded by the GABRA1 gene): K401fs (c.1200del), S326fs (c.975del), V290fs (c.869_888del), and F272fs (c.813del). These variants result in the loss of one to three transmembrane helices, whereas wild type 1 has four transmembrane helices. Therefore, these variants serve as valuable models to evaluate membrane protein biogenesis and proteostasis deficiencies of GABAARs. In HEK293T cells, all four frameshift variants exhibit significantly reduced trafficking to the cell surface, resulting in essentially non-functional ion channels. However, the severity of proteostasis deficiency varied among these four frameshift variants, presumably due to their specific transmembrane domain deletions. The variant 1subunits exhibited endoplasmic reticulum (ER) retention and activated the unfolded protein response (UPR) to varying extents. Our findings revealed that these frameshift variants of GABRA1 utilize overlapping yet distinct molecular mechanisms to impair proteostasis, providing insights into the pathogenesis of GABAAR-associated epilepsy.

cell biology↗

bioRxiv2023

Pathogenicity Prediction of GABAA Receptor Missense Variants

Variants in the genes encoding the subunits of gamma-aminobutyric acid type A (GABAA) receptors 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 for GABAA receptor variants. Here, we apply two state-of-the-art modeling tools, namely AlphaMissense, which uses an artificial intelligence-based approach based on AlphaFold structures, and Rhapsody, which integrates sequence evolution and known structure-based data, 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. Our results demonstrate that the predicted pathogenicity correlates well between AlphaMissense and Rhapsody although AlphaMissense tends to generate higher pathogenic probability. Furthermore, almost all annotated pathogenic variants in the ClinVar clinical 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.

biochemistry↗

bioRxiv2023

Synchrony of Bird Migration with Avian Influenza Global Spread; Implications for Vulnerable Bird Orders

Highly pathogenic avian influenza virus (HPAIV) A H5 clade 2.3.4.4 has caused worldwide outbreaks in domestic poultry, occasional spillover to humans, and increasing deaths of diverse species of wild birds since 2014. Wild bird migration is currently acknowledged as an important ecological process contributing to the global dispersal of HPAIV H5. However, it is unclear how seasonal bird migration facilitates global virus dispersal, and which avian species are exposed to HPAI H5 clade 2.3.4.4 viruses and where. To shed light on ongoing global outbreaks, we sought to explore these questions through phylodynamic analyses based on empirical data of bird movement tracking and virus genome sequences. First, based on viral phylogeography and bird migration networks, we demonstrate that seasonal bird migration can explain salient features of the global dispersal of clade 2.3.4.4. Second, we detect synchrony between the seasonality of bird annual cycle phases and virus lineage movements. We reveal the differing vulnerable bird orders at geographical origins and destinations of HPAIV H5 lineage movements. Notably, we highlight the potential importance of relatively under-discussed Suliformes and Ciconiiformes, in addition to Anseriformes and Charadriiformes, in virus lineage movements. Our study provides a phylodynamic framework that links the bird movement ecology and genomic epidemiology of avian influenza; it highlights the importance of integrating bird behaviour and life history in avian influenza studies.

ecology↗

bioRxiv2023

Adapting the endoplasmic reticulum proteostasis rescues epilepsy-associated NMDA receptor variants

The GRIN genes encoding N-methyl-D-aspartate receptor (NMDAR) subunits are remarkably intolerant to variation. Many pathogenic NMDAR variants result in their protein misfolding, inefficient assembly, reduced surface expression, and impaired functionality at the plasma membrane, causing neurological disorders including epilepsy and intellectual disability. Here, we concentrate on the proteostasis maintenance of NMDARs containing epilepsy-associated variations in the GluN2A (or NR2A) subunit, including M705V and A727T. We showed that these two variants are targeted to the proteasome for degradation and have reduced functional surface expression. We demonstrated that the application of BIX, a known small molecule activator of an HSP70 family chaperone BiP (Binding immunoglobulin Protein) in the endoplasmic reticulum (ER), significantly increases total and surface protein levels, and thus the function of the M705V and A727T variants in HEK293T cells. Mechanistic studies revealed that BIX promotes folding, inhibits degradation, and enhances anterograde trafficking of the M705V variant by modest activation of the IRE1 pathway of the unfolded protein response. Our results showed that adapting the ER proteostasis network restores the folding, trafficking, and function of pathogenic NMDAR variants, representing a potential treatment for neurological disorders resulting from NMDAR dysfunction.

cell biology↗

bioRxiv2022

Quantitative interactome proteomics identifies proteostasis network for GABAA receptors

Gamma-aminobutyric acid type A (GABAA) receptors, the primary inhibitory neurotransmitter-gated ion channels in the mammalian central nervous system, inhibit neuronal firing to preserve balanced neuronal activity. Maintenance of GABAA receptor protein homeostasis (proteostasis) in the cell utilizing its interacting proteins is essential for the function of GABAA receptors. However, how the proteostasis network orchestrates GABAA receptor biogenesis in the endoplasmic reticulum (ER) is not well understood. To address this question systematically, we employed a proteomics-based approach to identify the interactomes of GABAA receptors by carrying out a quantitative immunoprecipitation-tandem mass spectrometry (IP-MS/MS) analysis utilizing stable isotope labeling by amino acids in cell culture (SILAC). To enhance the coverage and reliability of the identified proteins, we performed comparative proteomics by using both wild type 1 subunit and a misfolding-prone 1 subunit carrying the A322D variant as the bait proteins. The wild type 1 interactome contains 125 proteins, the 1(A322D) interactome contains 105 proteins, and 54 proteins overlap within two interactomes. Bioinformatics analysis identified potential GABAA receptor proteostasis network components, including chaperones, folding enzymes, trafficking factors, and degradation factors. Further, their potential involvement is modelled in the cellular folding, degradation and trafficking pathways for GABAA receptors. In addition, we verified endogenous interactions between 1 subunit and their selected interactors by carrying out co-immunoprecipitation assay in mouse brain homogenates. This study paves the way for understanding the molecular mechanisms as well as fine-tuning of GABAA receptor proteostasis to ameliorate related neurological diseases such as epilepsy.

biochemistry↗

bioRxiv2019

Characterizing the Emergence of Liver and Gallbladder from the Embryonic Endoderm through Single-Cell RNA-Seq

The liver and gallbladder are among the most important internal organs derived from the endoderm. Several inductive signals regulate liver development, yet the pure nascent hepatic and gallbladder cells are unable to be isolated due to limited cell markers and cell numbers. The transcriptome networks of the hepatic lineage in the endoderm, and how the gallbladder differentiates from the adjacent endoderm population, are not fully understood. Using a transgenic Foxa2eGFP reporter mouse line, we performed deep single-cell RNA sequencing on 1,966 individual cells, including nascent hepatic and gallbladder cells, isolated from the endoderm and hepatic regions from ten embryonic stages, ranging from day E7.5 to E15.5. We identified the embryonic liver developmental trajectory from primitive streak to hepatoblasts and characterized the transcriptome of the hepatic lineage. During pre-hepatogenesis (5-6 somite stage), we have identified two groups of foregut endoderm cells, one derived from visceral endoderm and the second derived from primitive streak via a mesenchymal-epithelial transition (MET). During the liver specification stages, liver primordium was identified to share both foregut and liver features. We also documented dynamic gene expression during the epithelial-hepatic transition (EHT). Six gene groups were found to switch on or off at different stages during liver specification. Importantly, we found that RXR complex signaling and newly identified transcription factors associated with liver specification. Moreover, we revealed the gallbladder primordium cells at E9.5 and found genes that transcriptionally distinguish them from the liver primordium. The present data provides a high-resolution resource and critical insights for understanding the emergence of the endoderm, liver and gallbladder development.

developmental biology↗