Biology subjects

Choi, Y. J.

Publications and source records attributed to Choi, Y. J..

9 recordsLinked to original sources

bioRxiv2026

TDP-43 amyloid conversion in cells tracks the net charge of a polyampholyte motif, not condensate solidification

Liquid-liquid phase separation of TDP-43 is implicated in ALS and FTLD, yet how its condensates convert into pathological amyloid in living cells remains poorly defined. Here we identify a polyampholyte motif (PAM) in the linker between RRM2 and the amyloid-forming low-complexity domain as a charge-tunable determinant of fibrillization that operates independently of condensate solidification. In budding yeast expressing GFP-TDP-43{Delta}NLS, charge reversal of three arginines (R268E/R272E/R275E; 3R-E) converted liquid puncta into ProteoStat-positive fibrils. Unexpectedly, fluorescence recovery after photobleaching showed 3R-E condensates to be more mobile than those of wild type, whereas the least mobile condensates, in yeast and in human U2OS cells, remained ProteoStat-negative. Across 24 PAM charge variants, the proportion of fibril-bearing cells scaled linearly with net charge regardless of residue identity, with charge neutralization delaying rather than abolishing conversion. Phosphomimetic substitution of the PAM serines alone induced fibrils, implicating phosphorylation as a potential physiological modulator of PAM charge.

biochemistry↗

bioRxiv2025

Urolithin A Restores Mitochondrial Function and Reverses Cardiac Remodeling in Heart Failure with Preserved Ejection Fraction

BackgroundHeart failure with preserved ejection fraction (HFpEF) accounts for nearly half of all heart failure cases; however, mechanism-based therapies targeting cellular dysfunction remain absent. Emerging evidence suggests that mitochondrial dysfunction and impaired quality control, particularly defective mitophagy, are central to the pathogenesis of HFpEF. Urolithin A (UA), a gut microbiome-derived postbiotic metabolite, has shown promise as a mitophagy activator in preclinical models; however, its therapeutic efficacy in HFpEF remains unknown. MethodsWe used a clinically relevant two-hit HFpEF mouse model (high-fat diet (HFD) plus N{omega}-nitro-L-arginine methyl ester (L-NAME)) and co-administered UA during disease progression. The cardiac structure and function were assessed using echocardiography, histology, and transmission electron microscopy. Mitochondrial bioenergetics were evaluated using Seahorse-based respirometry. Mitophagy flux was monitored using mt-Keima assays in H9c2 cells and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Mechanistic insights were obtained using immunoblotting, integrated shotgun metagenomic and lipidomic profiling, and single-nucleus RNA sequencing (snRNA-seq). ResultsUA treatment significantly attenuated cardiac remodeling and fibrosis in HFpEF mice while improving diastolic function parameters. Transmission electron microscopy revealed restoration of the mitochondrial ultrastructure, and mitochondrial stress tests demonstrated enhanced oxidative phosphorylation capacity and glycolytic reserves. Immunoblot assays revealed that UA treatment recovered PINK1/Parkin-mediated mitophagy markers (reduced LC3-II and p62/SQSTM1). Mt-Keima assays confirmed enhanced mitophagic flux in H9c2 cells under HFpEF-like stress conditions. Integrated metagenomics and lipidomics revealed significant reductions in cardiovascular risk-associated ceramides. snRNA-seq demonstrated that HFpEF-like stress downregulated contractile, calcium-handling, and mitochondrial mitophagy gene programs. UA treatment restored the mitochondrial quality control signatures and normalized profibrotic and conduction-type cell populations. ConclusionsUrolithin A restored mitochondrial quality control through the activation of mitophagy and reversed cardiac remodeling in HFpEF. These findings establish UA as a mitochondria-targeted therapeutic candidate for HFpEF. Novelty and SignificanceO_ST_ABSWhat is known?C_ST_ABS{checkmark} Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome driven by metabolic stress and myocardial remodeling, for which effective disease-modifying therapies are lacking. {checkmark}Mitochondrial dysfunction and impaired mitophagy have been implicated in HFpEF, but their roles in cardiomyocyte state remodeling and fibrosis remain incompletely defined. {checkmark}Urolithin A, a gut microbiome-derived metabolite, enhances mitophagy in aging and metabolic tissues, but its relevance to HFpEF has not been established. What new information does this article contribute?{checkmark} Urolithin A restores mitochondrial quality control and mitophagic flux in cardiomyocytes, improving diastolic function and attenuating cardiac remodeling in a two-hit HFpEF model. {checkmark}Integrated metagenomic and lipidomic analyses identify suppression of gut microbiome-associated ceramide biosynthesis as a systemic mechanism linked to Urolithin A-mediated cardioprotection. {checkmark}Single-nucleus RNA sequencing reveals that Urolithin A reverses maladaptive cardiomyocyte transcriptional state transitions toward fibrogenic and conduction-like programs.

molecular biology↗

bioRxiv2025

Polyelectrolyte domains in intrinsically disordered regions differentially regulate intracellular biomolecular condensation

Intracellular condensation is a hallmark of many proteins containing intrinsically disordered regions (IDRs). Decoding the regulatory mechanisms of condensation encoded within the amino acid sequences of condensate-forming proteins is essential for understanding both how intracellular condensates assemble and how they contribute to various disease pathologies. Here, we investigated the budding yeast P-body proteins Nst1 and Edc3, which exhibit pronounced intracellular self-condensation, and identified charged amino acid clusters in the low complexity regions (LCRs) that modulate their phase behavior. Guided by the stickers-and-spacers framework, we hypothesized that clusters of charged residues within IDRs function in promoting intracellular condensation process. Using a sliding-window approach, we designated and dissected the polyelectrolyte (PE) domains of Nst1 and Edc3 and established their essential contributions to condensate coalescence. In Nst1, the polyanionic (PA) subdomain enhances intermolecular attraction and promotes coalescence, whereas the polyampholyte (PAM) subdomain imparts rigidity to the condensate as a distinct modulatory element and functions as a platform in condensation process of other P-body components. PA of Edc3 also regulates coalescence. Extending these findings to the neurodegenerative disease-associated protein TDP-43, we show that its PA regions are critical for coalescence, consistent with the presence of negatively charged amino acid clusters as potent molecular stickers functioning in condensate coalescence in cells. These results highlight charged-residue clusters as important sequence determinants of protein phase behavior and suggest that they represent promising novel regulatory targets for modulating TDP-43 condensation in cells.

cell biology↗

bioRxiv2025

Hypoosmolarity inhibits archaeal ammonia oxidation

Salinity strongly influences the physiology and distribution of nitrifying microorganisms, yet the effects of low salinity on them remain understudied. This study investigates the impact of hypoosmolarity on different groups of ammonia oxidizers in soil and lake environments, as well as in pure culture isolates. In soil microcosms amended with ammonium, at low salinity levels ([~]120 S/cm), comparable to values commonly found in pristine terrestrial and aquatic environments, the abundance of ammonia-oxidizing bacteria (AOB), dominated by Nitrosomonas oligotropha, significantly increased. In contrast, the growth of ammonia-oxidizing archaea (AOA), dominated by "Ca. Nitrosotenuis" of the Nitrosopumilaceae family, was stimulated by high salinity ([~]760 S/cm). In ammonium-fed lake microcosms, the abundance of AOB, dominated by N. oligotropha, significantly increased under both low ([~]170 S/cm) and high salinity ([~]850 S/cm) conditions. In the presence of allylthiourea, a bacterial nitrification inhibitor, AOA were sensitive to low salinity in both soil and lake microcosms. Consistently, pure culture studies revealed marked growth inhibition of AOA, especially members of Nitrosopumilaceae, under hypoosmolarity, unlike AOB and complete ammonia oxidizer (comammox) strains. Comparative genomic analyses with AOB and comammox, along with transcriptomic studies, suggested that the sensitivity of AOA to hypoosmolarity stress was possibly due to a lack of sophisticated osmoregulatory transport systems and their S-layer cell wall structure. Overall, this study highlights hypoosmolarity as a key factor shaping the ecological niches and distribution of ammonia oxidizers, as well as nitrification activities, in terrestrial and aquatic environments that are increasingly affected by intensified water cycles due to climate change.

microbiology↗

bioRxiv2024

Quantification of tissue creatine content using capillary electrophoresis

Creatine plays a fundamental role in cellular energy homeostasis. The current protocol describes an alternative method for creatine quantification in biological tissue samples using capillary electrophoresis, with high separation efficiency that enables rapid analysis with low sample volumes. The protocol involves homogenization of snap-frozen tissue in phosphate buffer, followed by electrophoresis through a bare-fused capillary (75 {micro}m internal diameter) and measurement at 200 nm on the Agilent 7100 CE system. Under the optimised conditions, there was excellent linearity in creatine standards between 6.3 - 100 {micro}M. The overall intra-assay variability for concentrations between 6.3 - 100 {micro}M was 1.5 %, and the inter-assay variability was 6.4 %, with a limit of detection at 6 nmol/mg protein. The protocol was further benchmarked against a commercially available enzyme assay kit using lung samples from lambs that received continuous creatine or saline supplementation. There was good agreement between the two methods (mean difference = 0.42 [-0.26-1.1] nmol/mg protein). Importantly, capillary electrophoresis enables reliable detection of creatine in biological samples from just [~]1.5 mg of wet-weight lung tissue. Capillary electrophoresis enables rapid (<10 minutes) and highly efficient analysis of tissue samples and avoids challenges faced with traditional enzymatic assays. The current protocol was developed and optimised with ovine lung tissue, but it can be easily adapted to analyse various tissue types. For tissues with higher baseline creatine content, such as the skeletal muscles or brain, <1 mg wet weight tissue would be sufficient to detect creatine using capillary electrophoresis.

biochemistry↗

bioRxiv2023

Structural and functional insights into the enzymatic plasticity of the SARS-CoV-2 NiRAN Domain

The enzymatic activity of the SARS-CoV-2 nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain is essential for viral propagation, with three distinct activities associated with modification of the nsp9 N-terminus, NMPylation, RNAylation, and deRNAylation/capping via a GDP-polyribonucleotidyltransferase reaction. The latter two activities comprise an unconventional mechanism for initiating viral RNA 5-cap formation, while the role of NMPylation is unclear. The structural mechanisms for these diverse enzymatic activities have not been properly delineated. Here we determine high-resolution cryo-electron microscopy structures of catalytic intermediates for the NMPylation and deRNAylation/capping reactions, revealing diverse nucleotide binding poses and divalent metal ion coordination sites to promote its repertoire of activities. The deRNAylation/capping structure explains why GDP is a preferred substrate for the capping reaction over GTP. Altogether, these findings enhance our understanding of the promiscuous coronaviral NiRAN domain, a therapeutic target, and provide an accurate structural platform for drug development.

biophysics↗

bioRxiv2021

Klf5 establishes bi-potential cell fate by dual regulation of ICM and TE specification genes

Early blastomeres of mouse preimplantation embryos exhibit bi-potential cell fate, capable of generating both embryonic and extra-embryonic lineages in blastocysts. Here, we identified three major 2 cell (2C) specific endogenous retroviruses (ERVs) as the molecular hallmark of the bi-potential plasticity. Using the LTRs of all three 2C-ERVs, we identified Klf5 as their major upstream regulator. Klf5 is essential for bi-potential cell fate: a single Klf5-overexpressing ESC generated terminally differentiated embryonic and extra-embryonic lineages in chimeric embryos, and Klf5 directly induces both ICM and TE specification genes. Intriguingly, Klf5 and Klf4 act redundantly during ICM specification, whereas Klf5 deficiency alone impairs TE specification. Klf5 is regulated by multiple 2C-specific transcription factors, particularly Dux, and the Dux/Klf5 axis is evolutionarily conserved. Altogether, the 2C-specific transcription program converges on Klf5 to establish bi-potential cell fate, enabling a cell state with dual activation of ICM and TE genes.

developmental biology↗

bioRxiv2021

Structural basis for backtracking by the SARS-CoV-2 replication-transcription complex

Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein crosslinking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3-segment of the product-RNA generated by backtracking extrudes through the RdRp NTP-entry tunnel, that a mismatched nucleotide at the product-RNA 3-end frays and enters the NTP-entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals. Significance StatementThe COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 genome is replicated and transcribed by its RNA-dependent RNA polymerase (RdRp), which is the target for antivirals such as remdesivir. We use a combination of approaches to show that backtracking (backwards motion of the RdRp on the template RNA) is a feature of SARS-CoV-2 replication/transcription. Backtracking may play a critical role in proofreading, a crucial process for SARS-CoV-2 resistance against many antivirals.

biochemistry↗

bioRxiv2018

Development of three-dimensional articular cartilage construct using silica nano-patterned substrate

Current strategies for cartilage cell therapy are mostly based on the use of autologous chondrocytes or mesenchymal stem cells (MSCs). However, these cells have limitations of a small number of cells available and of low chondrogenic ability, respectively. Many studies now suggest that fetal stem cells are more plastic than adult stem cells and can therefore more efficiently differentiate into target tissues. This study introduces, efficiency chondrogenic differentiation of fetal cartilage-derived progenitor cells (FCPCs) to adult cells can be achieved using a three-dimensional (3D) spheroid culture method based on silica nanopatterning techniques. In evaluating the issue of silica nano-particle size (Diameter of 300, 750, 1200 nm), each particle size was coated into the well of a 6-well tissue culture plate. FCPCs (2 x 105 cells/well in 6-well plate) were seeded in each well with chondrogenic medium. In this studys, the 300 nm substrate that formed multi-spheroids and the 1200 nm substrate that showed spreading were due to the cell-cell adhesion force(via N-cadherin) and cell-substrate(via Integrin) force, the 750 nm substrate that formed the mass-aggregation can be interpreted as the result of cell monolayer formation through cell-substrate force followed by cell-cell contact force contraction. We conclude that our 3D spheroid culture system contributes to an optimization for efficient differentiation of FCPC, offers insight into the mechanism of efficient differentiation of engineered 3D culture system, and has promise for wide applications in regeneration medicine and drug discovery fields.

cell biology↗