Biology subjects

Chen, T.-Y.

Publications and source records attributed to Chen, T.-Y..

11 recordsLinked to original sources

bioRxiv2025

Conformation-gated binding drives negative cooperativity in ATP:cob(I)alamin Adenosyltransferase for optimized cobalamin handling

Vitamin B12 (cobalamin) is a high-value yet scarce cofactor required for various metabolic processes, making its efficient handling important for maintaining metabolic homeostasis. While the involvement of ATP:cob(I)alamin adenosyltransferases (MMAB) in the synthesis, delivery, and repair of 5-deoxyadenosylcobalamin (AdoCbl) is well established, the kinetic mechanisms that regulate this process, particularly its negative cooperativity, remain poorly understood. Understanding these mechanisms is key to clarifying how MMAB efficiently uses AdoCbl, prevents resource wastage, and supports bacterial survival in nutrient-limited environments. Using single-molecule relative fluorescence (SRF) spectroscopy, we found that conformation-gated binding is the driving force behind MMABs preference for AdoCbl over hydroxocobalamin and is the underlying mechanism for negative cooperativity. This mechanism significantly slows down the binding of the second equivalent of AdoCbl, favoring the singly bound state. Our findings indicate that MMAB predominantly binds a single AdoCbl, optimizing the AdoCbl loading to methylmalonyl-CoA mutase. Additionally, our SRF approach also serves as a tool to explore other cofactor interactions, such as those between riboswitches and cobalamin derivatives, to provide insights into regulatory mechanisms of cobalamin sensing and gene regulation, which are crucial for bacterial adaptation to changing nutrient conditions. Significance StatementMMAB is important for B12-dependent propionate metabolism in bacteria. Our findings reveal that conformation-driven binding mechanism underlines the negative cooperativity of MMAB, as it favors the binding of the first AdoCbl while limiting further binding. The larger kon for the first site, combined with similar unbinding rates for both sites, could provide a solution for optimizing cobalamin handling and minimize unnecessary waste. Our single-molecule fluorescence approach offers a powerful tool for investigating other dynamic cofactor interactions, providing new insights into regulatory mechanisms in bacterial metabolism.

biophysics↗

bioRxiv2024

Guard-cell phytosterol homeostasis is critical for proper stomatal development

Stomata regulate gas exchange and control water loss in response to the environmental stimuli and their distribution in the leaf epidermis is tightly regulated during development to ensure proper patterns. Although many studies have focused on the function of early stomatal lineage cells, little is known about the role of mature guard cells (GCs) in maintaining stomatal distribution. Here, we identified a previously uncharacterized enzyme, GDSL-type sterol esterase (GSEase), that is specifically expressed in mature guard cells and catalyzes lipid droplet-stored phytosterol ester degradation. Loss of GSEase decreased the level of free campesterol, a biosynthetic precursor of brassinosteroids (BRs), reduced BR level, and increased stomatal density in leaves, which could be further rescued by increasing the BR signaling. Furthermore, selectively reducing the BR response in GCs by utilizing the GSEase promoter-driven det2-1, a mutation causing BR biosynthesis deficiency, resulted in an elevated stomatal count, as demonstrated in gsease plants. These results indicate that GSEase plays a critical role in maintaining phytosterol homeostasis in GCs and the released phytosterols suppress the initiation of stomatal development in adjacent cells though the BR pathway.

plant biology↗

bioRxiv2022

Assessment of mosquito longevity, fecundity, and dengue virus titer following exposure to plasma from a host with Type 2 diabetes mellitus - a pilot study

The mosquito Aedes aegypti (Linnaeus) prefers feeding on humans and will encounter many blood components that might influence mosquito physiology and vectorial capacity. The effects of dengue virus infection in patients with type-2 diabetes mellitus revealed an influence on platelet numbers in blood and diabetic persons are more likely to develop severe dengue. Infection with dengue virus (DENV) is known to impact diabetic patients but the impact of diabetic blood on mosquito vectorial capacity is not known. This pilot study investigated the effects of ingesting blood plasma from a patient with diabetes on mosquito fecundity, longevity, and vector competence for DENV-2 in the mosquito, Ae. aegypti. Female mosquitoes fed on plasma from a diabetic individual laid significantly more eggs. Diabetic plasma shortened overall mosquito life span. Vector competence was significantly different between extrinsic incubation periods at 7 (3.44{+/-}2.5 log PFUe DENV) and 14 days post-infection (4.28{+/-}2.4 log PFUe DENV) in mosquitoes fed DENV-2 in plasma from a diabetic individual. As this study is an initial investigation into the influence of diabetes plasma on mosquito life history traits, a more comprehensive study with more samples of diabetes plasma is underway and should yield more details about the impact of diabetes on mosquito biology.

ecology↗

bioRxiv2022

Subcellular Redox Responses Reveal Different Cu-dependent Antioxidant Defenses between Mitochondria and Cytosol

Excess intracellular Cu perturbs cellular redox balance and thus causes diseases. However, the relationship between cellular redox status and Cu homeostasis and how such an interplay is coordinated within cellular compartments has not yet been well established. Using combined approaches of organelle-specific redox sensor Grx1-roGFP2 and non-targeted proteomics, we investigate the real-time Cu-dependent antioxidant defenses of mitochondria and cytosol in live HEK293 cells. The Cu-dependent real-time imaging experiments show that CuCl2 treatment results in increased oxidative stress in both cytosol and mitochondria. In contrast, subsequent Cu depletion by BCS, a Cu chelating reagent, lowers oxidative stress in mitochondria but causes even higher oxidative stress in the cytosol. The proteomic data reveal that several mitochondrial proteins, but not cytosolic ones, undergo significant abundance change under Cu treatments. The proteomic analysis also shows that proteins with significant changes are related to mitochondrial oxidative phosphorylation and glutathione synthesis. The differences in redox behaviors and protein profiles in different cellular compartments reveal distinct mitochondrial and cytosolic response mechanisms upon Cu-induced oxidative stress. These findings provide insights into how redox and Cu homeostasis interplay by modulating specific protein expressions at the subcellular levels, shedding light on understanding the effects of Cu-induced redox misregulation on the diseases. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/500983v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@849766org.highwire.dtl.DTLVardef@efb723org.highwire.dtl.DTLVardef@1e097d5org.highwire.dtl.DTLVardef@105c62_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Identification of a distal RXFP1 gene enhancer with differential activity in fibrotic lung fibroblasts involving AP-1

Relaxin/insulin-like family peptide receptor 1 (RXFP1) mediates relaxins antifibrotic effects and has reduced expression in the lung and skin of patients with fibrotic interstitial lung disease (fILD) including idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc). This may explain the failure of relaxin-based anti-fibrotic treatments in SSc, but the regulatory mechanisms controlling RXFP1 expression remain largely unknown. This study aimed to identify regulatory elements of RXFP1 that may function differentially in fibrotic fibroblasts. We identified and evaluated a distal regulatory region of RXFP1 in lung fibroblasts using a luciferase reporter system. Using serial deletions, an enhancer upregulating pGL3-promoter activity was localized to the distal region between -584 to -242bp from the distal transcription start site (TSS). This enhancer exhibited reduced activity in IPF and SSc lung fibroblasts. Bioinformatic analysis identified two clusters of activator protein 1 (AP-1) transcription factor binding sites within the enhancer. Site-directed mutagenesis of the binding sites confirmed that only one cluster reduced activity (-358 to -353 relative to distal TSS). Co-expression of FOS in lung fibroblasts further increased enhancer activity. In vitro complex formation with a labeled probe spanning the functional AP-1 site using nuclear proteins isolated from lung fibroblasts confirmed a specific DNA/protein complex formation. Application of antibodies against JUN and FOS resulted in the complex alteration, while antibodies to JUNB and FOSL1 did not. Analysis of AP-1 binding in 5 pairs of control and IPF lung fibroblasts detected positive binding more frequently in control fibroblasts. Expression of JUN and FOS was reduced and correlated positively with RXFP1 expression in IPF lungs. In conclusion, we identified a distal enhancer of RXFP1 with differential activity in fibrotic lung fibroblasts involving AP-1 transcription factors. Our study provides insight into RXFP1 downregulation in fILD and may support efforts to reevaluate relaxin-based therapeutics alongside upregulation of RXFP1 transcription.

molecular biology↗

bioRxiv2021

High CXCL6 drives matrix expression and correlate with markers of poor outcome in IPF

Signaling via G protein-coupled receptors (GPCRs) can modulate levels of cyclic adenosine monophosphate (cAMP) and shape the functions of fibroblasts in idiopathic pulmonary fibrosis (IPF). We have identified Chemokine (C-X-C) Motif Ligand 6 (CXCL6) as a potential pro-fibrotic GPCR ligand. We tested the function of CXCL6 in ex vivo human donor and fibrotic lung fibroblasts and in an animal model of pulmonary fibrosis. We also measured levels of CXCL6 in the blood and bronchoalveolar lavage (BAL) of patients with IPF. CXCL6 decreased cAMP levels in a dose-dependent manner in Donor and IPF Fibroblasts. CXCL6 mRNA and protein were localized to epithelial cells. Administration of mCXCL5 (LIX, murine CXCL6 homologue) to mice increased collagen synthesis with and without bleomycin. CXCL6 increased Collagen I and -SMA levels in Donor and IPF Fibroblasts. Silencing of CXCR1/2 as well as Reparixin, a CXCR1/2 inhibitor, blocked effects of CXCL6. Treprostinil blocked effects of CXCL6 only on levels of -SMA but not on Collagen I. CXCL6 levels in the BAL of two separate cohorts of patients with IPF was associated with poor survival. We conclude that high CXCL6 drives fibroblast function and correlates with poor outcomes in IPF.

cell biology↗

bioRxiv2021

Activation of the autophagy pathway affects Dengue virus infection in Aedes aegypti

Mosquito-borne Dengue virus (DENV) has caused major disease worldwide, impacting 50 to 100 million people every year, and is spread by the major mosquito vector Aedes aegypti. Understanding mosquito physiology and developing new control strategies becomes an important issue to eliminate DENV. We focused on autophagy, a pathway suggested as having a positive influence on virus replication in humans, as a potential anti-viral target in the mosquito. To understand the role played by autophagy in Ae. aegypti, we examined the expression of the pathway in vitro (Aag-2 cell) and in vivo (Ae. aegypti). The results indicated that DENV infection in Aag-2 cells caused the microtubule-associated protein light chain 3-phosphatidylethanolamine conjugate (LC3-II) protein levels to increase which indicated the activation of the autophagy pathway. Rapamycin and 3-Methyladenine were used to activate or suppress the autophagy pathway, respectively. Rapamycin treatment decreased the virus titer in the Aag-2 cells, but the 3-Methyladenine treatment did not affect DENV titer. In Ae. aegypti, microinjected rapamycin increased the DENV titer after one-day infection and was significantly different compared to the control group titer. Two ATG genes, ATG4 and ATG12, were expressed differentially under the rapamycin treatments. Although the results differed between in vitro and in vivo studies, findings from both support the interaction between autophagy and DENV. Our studies revealed the activation of the autophagy pathway through rapamycin could be related to DENV infection in the mosquito. The possibility of autophagy being associated with different antiviral mechanisms at different extrinsic incubation times and tissues in Ae. aegypti is discussed. Author SummaryDengue virus (DENV) has been a great threat to public health and has not developed an efficient method to stop the transmission. To understand the complex interaction between virus and mosquito, we investigate the autophagy pathway and its role during the infection process. We noticed the induction of autophagy pathways from DENV infection in Aag-2 cells and blood meal from Ae. aegypti. Moreover, activation of the autophagy pathway from rapamycin could alter the DENV titer. Our results indicated the autophagy pathway is associated with DENV and could be crucial during the DENV infection. Furthermore, we proved the practicality of small molecules in altering the autophagy pathway in mosquitoes, and thus the usage of small molecules as possible mosquito pathogen vaccines should be evaluated.

molecular biology↗

bioRxiv2020

Proton-Dependent Inhibition, Inverted Voltage Activation, and Slow Gating of CLC-0 Chloride Channel

CLC-0, a prototype Cl- channel in the CLC family, employs two gating mechanisms that control its ion-permeation pore: fast gating and slow gating. The negatively-charged sidechain of a pore glutamate residue, E166, is known to be the fast gate, and the swinging of this sidechain opens or closes the pore of CLC-0 on the millisecond time scale. The other gating mechanism, slow gating, operates with much slower kinetics in the range of seconds to tens or even hundreds of seconds, and it is thought to involve still-unknown conformational rearrangements. Here, we find that low intracellular pH (pHi) facilitates the closure of the CLC-0s slow gate, thus generating current inhibition. The rate of low pHi-induced current inhibition increases with intracellular H+ concentration ([H+]i)--the time constants of current inhibition by low pHi = 4.5, 5.5 and 6 are roughly 0.1, 1 and 10 sec, respectively, at room temperature. In comparison, the time constant of the slow gate closure at pHi = 7.4 at room temperature is hundreds of seconds. The inhibition by low pHi is significantly less prominent in mutants favoring the slow-gate open state (such as C212S and Y512A), further supporting the fact that intracellular H+ enhances the slow-gate closure in CLC-0. A fast inhibition by low pHi causes an apparent inverted voltage-dependent activation in the wild-type CLC-0, a behavior similar to those in some channel mutants such as V490W in which only membrane hyperpolarization can open the channel. Interestingly, when V490W mutation is constructed in the background of C212S or Y512A mutation, the inverted voltage-dependent activation disappears. We propose that the slow kinetics of CLC-0s slow-gate closure may be due to low [H+]i rather than due to the proposed large conformational change of the channel protein. Our results also suggest that the inverted voltage-dependent opening observed in some mutant channels may result from fast closure of the slow gate by the mutations.

biophysics↗

bioRxiv2020

Combination of kdr mutation and detoxification gene expression associated with high tolerance for permethrin in a resistant Aedes aegypti population

Aedes aegypti, as one of the vectors transmitting several arboviruses, is a main target in mosquito control programs. Permethrin remains the major adulticide used to control these mosquitoes. The increasing percentage of permethrin resistant Aedes aegypti has become an important issue around the world. Knockdown resistance (kdr) is one of the major mechanisms related to permethrin resistance. On the other hand, detoxification genes including cytochrome P450 monooxygenases (P450) and glutathione S-transferases (GSTs) are also suggested as permethrin resistance apparatus. Here we selected a permethrin resistant (p-s) Aedes aegypti population from Florida and compared its mortality after exposure, median lethal dose (LD50), adult survivorship and larval development to several field populations. We used allele-specific PCR genotyping of the S989P, V1016I and F1534C sites in the sodium channel gene and gene expression analysis of several p450 and GSTs genes before and after permethrin exposure to determine their involvement in permethrin sensitivity between Ae. aegypti populations. Results indicated the p-s population had the highest resistance to permethrin based on LD50 and the mortality test. The larval development time did not significantly differ between the populations, however the p-s adults survived longer than the other populations. In the genotype study, p-s population had mostly homozygous mutations in all three mutant sites of the sodium channel gene. Detoxification gene expression studies showed that two p450 genes, AAEL009124 (CYP6N12) and, AAEL009127 (CYP6M11), were upregulated and, accession # AAEL006802, AAEL014891 (CYP6P12) and AAEL014619 (CYP9J22) were downregulated after 120 minutes of permethrin exposure in the p-s population. These results suggest that in highly permethrin resistant Aedes aegypti populations both kdr mutations and xenobiotic metabolism might be involved. Involvement of multiple mechanisms to achieve resistance to permethrin supports the need for implementing comprehensive mosquito control measures, such as an integrated pest management strategy, so that selection pressure for resistance is decreased without compromising control efforts while new methodologies are being developed. Author summaryPyrethroids have been applied as a major type of insecticide targeted at Aedes aegypti, a key vector in the transmission of several flaviviruses. Resistance to pyrethroids has emerged and has become a worldwide threat to mosquito control. Pyrethroid resistance usually occurs with knockdown resistance (kdr) where the voltage gated sodium channel is mutated. We selected a permethrin resistant (p-s) Aedes aegypti population from Florida and, along with two other field populations, examined three mutation sites, S989P, V1016I and F1534C. The data showed the p-s population had the most homozygous mutations which correlated to the permethrin resistance level. Besides kdr, detoxification genes also have been identified to have pyrethroid metabolizing abilities. We found two cytochrome P450 monooxygenases genes, CYP6N12 and CYP6M11, were overexpressed in the p-s population after permethrin exposure, suggesting a role in resistance to permethrin. Together, our results provide information about potential mechanisms used in major vectors with high insecticide resistance.

molecular biology↗

bioRxiv2018

Super-enhancer impairment is a link between MLL4 inactivated lung tumors and their vulnerability to glycolysis pathway inhibition

Epigenetic modifiers often harbor loss-of-function mutations in lung cancer, but their tumor-suppressive roles are poorly characterized. Here we show that lung-specific loss of the gene encoding the histone methyltransferase MLL4 (alias KMT2D; a COMPASS-like enzyme), which is ranked the most highly inactivated epigenetic modifier in lung cancer, strongly promotes lung adenocarcinoma in mice. Mll4 loss upregulated tumor-promoting programs, including glycolysis. The pharmacological inhibition of glycolysis preferentially impeded tumorigenic growth of human lung cancer cells bearing MLL4-inactivating mutations. Mll4 loss widely impaired epigenomic signals for super-enhancers and enhancers, including the super-enhancer for the circadian rhythm repressor gene Per2, and decreased Per2 expression. Per2 downregulated several glycolytic pathway genes. These findings uncover a distinct tumor-suppressive epigenetic mechanism in which MLL4 enhances Per2-mediated repression of pro-tumorigenic glycolytic genes via super-enhancer activation to suppress lung adenocarcinoma tumorigenesis and also implicate a glycolysis-targeting strategy as a therapeutic intervention for the treatment of MLL4- mutant lung cancer.

cancer biology↗

bioRxiv2018

Peripheral, intrareceptor inhibition in mosquito olfaction

How chemical signals are integrated at the peripheral sensory system of insects is still an enigma. Here we show that when coexpressed with Orco in Xenopus oocytes, an odorant receptor from the southern house mosquito, CquiOR32, generated inward (regular) currents when challenged with cyclohexanone and methyl salicylate, whereas eucalyptol and fenchone elicited inhibitory (upward) currents. Responses of CquiOR32-CquiOrco-expressing oocytes to odorants were reduced in a dose-dependent fashion by coapplication of inhibitors. This intrareceptor inhibition was also manifested in vivo in fruit flies expressing the mosquito receptor CquiOR32, as well in neurons on the antennae of the southern house mosquito. Likewise, an orthologue from the yellow fever mosquito, AaegOR71, showed intrareceptor inhibition in the Xenopus oocyte recording system and corresponding inhibition in antennal neurons. Intrareceptor inhibition was also manifested in mosquito behavior. Blood-seeking females were repelled by methyl salicylate, but repellence was significantly reduced when methyl salicylate was coapplied with eucalyptol.\n\nOne Sentence SummaryIntrareceptor inhibition was observed in mosquito odorant receptors expressed in heterologous systems, in vivo, and manifested in behavioral responses.

neuroscience↗