Biology subjects

Choi, T.

Publications and source records attributed to Choi, T..

9 recordsLinked to original sources

bioRxiv2025

Structural insights into how metallochaperones UreE and UreG interact to deliver a toxic metal to urease

Maturation of urease, a virulence factor for Helicobacter pylori infection, requires the delivery of nickel ions to the metalloenzyme. To avoid cytotoxicity, nickel ions are transferred within protein complexes of metallochaperones to ensure that the toxic metal do not escape into the cytoplasm. In the urease maturation pathway, metallochaperone UreG receives its nickel ions by forming a 2:2 complex with another metallochaperone UreE. Using C-terminal truncation variants of UreE [UreE(1-158) and UreE(1-148)], we determined the crystal structures of the UreE2G2 complex bound with nickel ions and GMPPNP, a non-hydrolysable GTP analogue. UreE-UreG interactions are asymmetric, where the UreG dimer mainly interacts with the proximal UreE. GTP binding induces conformational changes in the G2 and CPH motifs of distal UreG that enable its anchorage to the UreE2G2 complex. His68 of distal UreG moves towards and chelates a nickel ion at the UreE binding site. Formation of the UreE2G2 complex juxtaposes the nickel binding sites of UreE and UreG. Nickel transfer from UreE to UreG induces conformational changes that weaken UreE-UreG interactions, thereby facilitating UreG dissociation. We further demonstrated that the hydrogenase maturation factor HypA, providing the nickel source, could activate urease in vitro through protein-protein interaction with wild-type UreE but not with its truncation variants. UreG binding induces conformational changes in the C-terminal tail of UreE, promoting dissociation of HypA-UreE complex. Our work presents a paradigm on how GTP and nicking binding allosterically regulate the formation of a metallochaperone complex to facilitate nickel transfer. SignificanceOur work provides insights into how cells solve the problem of trafficking a toxic metal, nickel, to the active site of urease. Colonization of Helicobacter pylori in acidic human stomach requires the biosynthesis of active urease, which involves the delivery of the toxic nickel ions to the active site of the metalloenzyme. To avoid cytotoxicity, nickel ions are transported from one metallochaperone to another via the formation of protein complexes so that the toxic metal ions do not escape into the cytoplasm. Supported by structural and biochemical evidence, we present a paradigm on how GTP and nickel binding allosterically promote the formation of a complex between metallochaperones UreE and UreG to facilitate nickel transfer between the two proteins.

biochemistry↗

bioRxiv2025

Restoration of Spermatogenesis is Dependent on Activation of a SPRY4-ERK Checkpoint Following Germline Stem Cell Damage

Mammalian spermatogonial stem cells (SSCs) sustain male fertility through continuous self-renewal and differentiation, leading to the production of haploid spermatozoa throughout adulthood. However, SSCs are vulnerable to genotoxic drugs, and patients receiving chemotherapy face a high risk of germline instability and infertility. The molecular mechanisms and cellular pathways that choreograph SSC recovery after chemotherapeutic insult remain unknown. Previously, we identified SPRY4 as an ERK-dependent negative feedback regulator of growth factor signaling that is critical for preservation of stem cell activity in cultured mouse SSCs. Here, we demonstrate that following alkylating agent busulfan (BU)-induced injury in adult mice, germline-specific Spry4 gene deletion (Spry4G-KO) reduces stem cell regeneration with an enhanced genotoxic stress response and differentiation with rapidly enhanced nuclear ERK1/2 activity in undifferentiated (Aundiff) spermatogonia (including SSCs). Genes essential for stem cell maintenance, including Id1 and Cxcl12, were dysregulated by loss of Spry4. Furthermore, the MEK1/2 inhibitor PD0325901, but not mTORC1 inhibitor Rapamycin, was sufficient to promote spermatogonial proliferation in Spry4G-KO testis 10 days post-BU treatment. Notably, the restoration of both spermatogonia pool and fertility was delayed in adult Spry4G-KO males long-term after injury. In summary, germline-specific deletion of Spry4 results in hyper-activation of the MAPK/ERK pathway in Aundiff spermatogonia, reducing spermatogonial genome integrity, unleashing excessive spermatogenesis after germline damage, and ultimately impairing germline regeneration in adult males. Our study indicates an essential role for SPRY4-ERK signaling as a molecular checkpoint in securing SSC recovery upon chemotherapy drug-induced germline damage, revealing how stem cells normally withstand environmental stress.

cell biology↗

bioRxiv2025

Cross cell-type systems genetics reveals the influence of eQTL at multiple points in the developmental trajectory of mouse neural progenitor cells

Genetic variation leads to phenotypic variability in pluripotent stem cells that presents challenges for regenerative medicine. Although recent studies have investigated the impact of genetic variation on pluripotency maintenance and differentiation capacity, less is known about how genetic variants affecting the pluripotent state influence gene regulation in later stages of development. Here, we characterized expression of more than 12,000 genes in 127 donor-matched Diversity Outbred (DO) mouse embryonic stem cell (mESC) and neural progenitor cell (mNPC) lines. Quantitative trait locus (QTL) mapping identified 2,947 expression QTL (eQTL) unique to DO mNPCs and 1,113 eQTL observed in both mNPCs and mESCs with highly concordant allele effects. We mapped three eQTL hotspots on Chromosomes (Chrs) 1, 10, and 11 that were unique to mNPCs. Target genes of the Chr 1 hotspot were overrepresented for those involved in mRNA processing, DNA repair, chromatin organization, protein degradation, and cell cycle. Mediation analysis of the Chr 1 hotspot identified Rnf152 as the best candidate mediator expressed in mNPCs, while cross-cell type mediation using mESC gene expression along with partial correlation analysis strongly implicated genetic variant(s) affecting Pign expression in the mESC state as regulating the mNPC Chr 1 eQTL hotspot. Together these findings highlight that many local eQTL confer similar effects on gene expression in multiple cell states; distant eQTL in DO mNPCs are numerous and largely unique to that cell state, with many co-localizing to mNPC-specific hotspots; and mediation analysis across cell types suggests that expression of Pign early in development (mESCs) shapes the transcriptome of the more specialized mNPC state.

genetics↗

bioRxiv2024

Chromosome X Dosage Modulates Development of Aneuploidy in Genetically Diverse Mouse Embryonic Stem Cells

The genetic integrity of pluripotent stem cells (PSC) is critical to their applications in research and therapy, but it is compromised by frequent development of structural chromosome variants associated with malignancy. Many cell lines exhibit remarkable genetic stability, but little is known about the basis of the known variation in genomic integrity amongst different PSC isolates. Here we identify aneuploidies using RNA-seq and proteomics data from a panel of mouse embryonic stem cell (mESC) lines derived from 170 Diversity Outbred mice. We found 62 lines with detectable aneuploid subpopulations and a subset of originally XX lines that lost one Chromosome X (XO). Strikingly, a much lower proportion of XX lines were aneuploid, compared to XY or XO lines. Two single-cell RNA-seq data sets demonstrated that aneuploid XY DO mESC also show lower Chromosome X gene expression, and a prospective study confirmed that XY mESC accumulate higher aneuploid proportions in culture than isogenic XX lines. We identify potential mechanisms for this protective effect of X chromosome dosage, including our findings that the lines with two active X Chromosomes have a higher proportion of 2-cell-like cells, a state associated with maintenance of genetic integrity of mESC, and that they show differential expression of X-linked tumor suppressor genes associated with the DNA damage response. HighlightsO_LIFirst genetic analysis of predisposition to aneuploidy in pluripotent stem cell cultures C_LIO_LIChromosomal regions duplicated in aneuploid mouse embryonic stem cells are syntenic with regions overrepresented in human pluripotent stem cell lines bearing recurrent genetic abnormalities C_LIO_LIX-Chromosome dosage strongly influences susceptibility to aneuploidy in mouse embryonic stem cells and to a lesser degree in human pluripotent stem cells C_LIO_LIXX mouse embryonic stem cell lines show a higher proportion of cells in 2 cell-like state and higher expression of tumor suppressor genes associated with DNA damage response C_LI

genetics↗

bioRxiv2023

3D modeling of CpG DNA binding with matrix lumican shows leucine-rich repeat motif involvement as in TLR9- CpG DNA interactions

Lumican is an extracellular matrix proteoglycan, known to regulate toll-like receptor (TLR) signaling in innate immune cells. In experimental settings, lumican suppresses TLR9 signaling by binding to, and sequestering its synthetic ligand, CpG-DNA, in non-signal permissive endosomes. However, the molecular details of lumican interactions with CpG-DNA are obscure. Here, the 3-D structure of the 22 base-long CpG-DNA (CpG ODN_2395) bound to lumican or TLR9 were modeled using homology modeling and docking methods. Some of the TLR9-CpG ODN_2395 features predicted by our model are consistent with the previously reported TLR9-CpG DNA crystal structure, substantiating our current analysis. Our modeling indicated a smaller buried surface area for lumican-CpG ODN_2395 (1803 A2) compared to that of TLR9-CpG ODN_2395 (2094 A2), implying a potentially lower binding strength for lumican and CpG-DNA than TLR9 and CpG-DNA. The docking analysis identified 32 amino acids in lumican LRR1-11 interacting with CpG ODN_2395, primarily through hydrogen bonding, salt-bridges and hydrophobic interactions. Our study provides molecular insights into lumican and CpG-DNA interactions that may lead to molecular targets for modulating TLR9 mediated inflammation and autoimmunity.

immunology↗

bioRxiv2022

Rebound Excitation of Epileptiform Activities by Transcranial Focused Ultrasound Stimulation

BackgroundConventional neurostimulations to treat epilepsy have adverse effects caused by post-inhibitory rebound excitations. Although ultrasound brain stimulation is feasible in inducing anticonvulsant effects, its association with paradoxical rebound excitations is unknown. ObjectiveThis study aimed at demonstrating rebound excitations with transcranial focused ultrasound. The modulations of epileptiform activities toward both suppressive and excitatory responses were investigated by changing ultrasonic transmit sequences. MethodsIn a pentylenetetrazol-injected acute epilepsy rat model, transcranial focused ultrasound stimulation was applied on the thalamus to modulate epileptiform activities. The parameters differentiated for pulse sequences were the pulse length, pulse pressure, and interval between the pulses. Sonication effects were assessed by electroencephalography (n=38), immuno-histochemical analysis (n=24), and optical measurement of cerebral blood volume changes (n=18). ResultsWhile ultrasonic patterns of stimuli at long intervals showed antiepileptic effects on electroencephalography, those at short intervals showed rebound excitatory responses followed by inhibitory activities. Further, suppressive states induced by inhibitory stimulations were transformed into excitatory states by applying a consecutive series of short bursts at higher acoustic pressure. Cerebral blood volume changes demonstrated consistent results with electroencephalography. Immunohistochemistry revealed that both inhibitory and excitatory neuronal cells were activated to generate rebound excitatory conditions, while inhibitory cells were activated for suppressive conditions. ConclusionsIn our study, variations in ultrasound stimulation patterns could modulate epileptiform activities in both upregulated and downregulated directions.

bioengineering↗

bioRxiv2022

Genetic dissection of the pluripotent proteome through multi-omics data integration

Genetic background is a major driver of phenotypic variability in pluripotent stem cells (PSCs). Most studies of variation in PSCs have relied on transcript abundance as the primary molecular readout of cell state. However, little is known about how proteins, the primary functional units in the cell, vary across genetically diverse PSCs, how protein abundance relates to variation in other cell characteristics, and how genetic background confers these effects. Here we present a comprehensive genetic study characterizing the pluripotent proteome of 190 unique mouse embryonic stem cell lines (mESCs) derived from genetically heterogeneous Diversity Outbred (DO) mice. The quantitative proteome is highly variable across DO mESCs, and we identified differentially activated pluripotency-associated pathways in the proteomics data that were not evident in transcriptome data from the same cell lines. Comparisons of protein abundance to transcript levels and chromatin accessibility show broad co-variation across molecular layers and variable correlation across samples, with some lines showing high and others low correlation between these multi-omics datasets. Integration of these three molecular data types using multi-omics factor analysis revealed shared and unique drivers of quantitative variation in pluripotency-associated pathways. QTL mapping localized the genetic drivers of this quantitative variation to a number of genomic hotspots, and demonstrated that multi-omics data integration consolidates the influence of genetic signals shared across molecular traits to increase QTL detection power and overcome the limitations inherent in mapping individual molecular features. This study reveals transcriptional and post-transcriptional mechanisms and genetic interactions that underlie quantitative variability in the pluripotent proteome, and in so doing provides a regulatory map for mouse ESCs that can provide a rational basis for future mechanistic studies, including studies of human PSCs.

genetics↗

bioRxiv2020

Using physiological signals to predict temporal defense responses: a multi-modality analysis

Defense responses are a highly conserved behavioral response set across species. Defense responses motivate organisms to detect and react to threats and potential danger as a precursor to anxiety. Accurate measurement of temporal defense responses is important for understanding clinical anxiety and mood disorders, such as post-traumatic stress disorder, obsessive compulsive disorder, and generalized anxiety disorder. Within these conditions, anxiety is defined as a state of prolonged defense response elicitation to a threat that is ambiguous or unspecific. In this study, we aimed to develop a data-driven approach to capture temporal defense response elicitation through a multi-modality data analysis of physiological signals, including electroencephalogram (EEG), electrocardiogram (ECG), and eye-tracking information. A fear conditioning paradigm was adopted to develop a defense response classification model. From a classification model based on 42 feature sets, a higher order crossing feature set-based model was chosen for further analysis with cross-validation loss of 0.0462 (SEM: 0.0077). To validate our model, we compared predicted defense response occurrence ratios from a comprehensive situation that generates defense responses by watching movie clips with fear awareness and threat existence predictability, which have been reported to correlate with defense response elicitation in previous studies. We observed that defense response occurrence ratios are correlated with threat existence predictability, but not with fear awareness. These results are similar to those of previous studies using comprehensive situations. Our study provides insight into measurement of temporal defense responses via a novel approach, which can improve understanding of anxiety and related clinical disorders for neurobiological and clinical researchers.

neuroscience↗

bioRxiv2019

Genetic variation influences pluripotent ground state stability in mouse embryonic stem cells through a hierarchy of molecular phenotypes

Mouse embryonic stem cells (mESCs) cultured under controlled conditions occupy a stable ground state where pluripotency-associated transcriptional and epigenetic circuitry are highly active. However, mESCs from some genetic backgrounds exhibit metastability, where ground state pluripotency is lost in the absence of ERK1/2 and GSK3 inhibition. We dissected the genetic basis of metastability by profiling gene expression and chromatin accessibility in 185 genetically heterogeneous mESCs. We mapped thousands of loci affecting chromatin accessibility and/or transcript abundance, including eleven instances where distant QTL co-localized in clusters. For one cluster we identified Lifr transcript abundance as the causal intermediate regulating 122 distant genes enriched for roles in maintenance of pluripotency. Joint mediation analysis implicated a single enhancer variant ~10kb upstream of Lifr that alters chromatin accessibility and precipitates a cascade of molecular events affecting maintenance of pluripotency. We validated this hypothesis using reciprocal allele swaps, revealing mechanistic details underlying variability in ground state metastability in mESCs.

genomics↗