Biology subjects

Mori, S.

Publications and source records attributed to Mori, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Characterization of a maintainable human myeloid model of VEXAS syndrome with enhanced TNF-induced cell death and DAMP release

Background: VEXAS syndrome is an adult-onset autoinflammatory disorder caused by somatic UBA1 mutations. UBA1-mutated myeloid cells have been reported to exhibit increased susceptibility to inflammatory cell death; however, the mechanisms underlying this phenotype and the extracellular consequences of enhanced cell death remain incompletely understood. Methods: Disease-associated UBA1 M41 mutations were introduced into U937 cells by CRISPR/Cas9-mediated genome editing. The resulting cells were characterized by genomic, molecular, and functional analyses, with particular focus on UBA1-associated cellular phenotypes, cell death signaling, and extracellular release of damage-associated molecular patterns (DAMPs). Results: Genome editing yielded UBA1-mutated cells that could be maintained under standard culture conditions while recapitulating multiple molecular and cellular features associated with VEXAS syndrome. Long-read sequencing revealed the intended UBA1 M41 mutation on one allele and a CRISPR/Cas9-induced on-target genomic deletion on the other. The mutant cells exhibited reduced UBA1b and increased UBA1c expression, prominent cytoplasmic vacuolization, reduced proliferative capacity, and increased basal cell death. They also showed enhanced susceptibility to TNF-induced cell death under conditions favoring either apoptosis or necroptosis. Mechanistic analyses demonstrated enhanced apoptotic and necroptotic signaling, together with increased basal abundance of RIPK1, RIPK3, and MLKL. Pharmacological inhibition of RIPK1, RIPK3, or MLKL attenuated membrane permeabilization under caspase-inhibited conditions. Enhanced cell death was accompanied by increased extracellular release of ATP, HMGB1, and S100A8/A9. Conclusions: We characterized a maintainable UBA1-mutated human myeloid model that recapitulates multiple molecular and cellular features associated with VEXAS syndrome. Using this model, we showed that UBA1 dysfunction is associated with heightened susceptibility to TNF-dependent apoptotic and necroptotic cell death and increased extracellular release of multiple DAMPs. These findings provide insight into the cellular consequences of UBA1 dysfunction and establish a tractable experimental platform for further mechanistic studies of VEXAS syndrome.

immunology↗

bioRxiv2026

TRPV1-cardiac afferent ablation after completed myocardial infarction prevents arrhythmogenic remodeling

Background Therapies to prevent ventricular arrhythmias after completed myocardial infarction (MI) remain limited. Although TRPV1 afferent ablation at acute MI improves cardiac remodeling, the effect of delayed subacute targeting remains unknown. Objective We investigated whether ablating cardiac TRPV1 afferents during the subacute post-MI window mitigates structural, electrophysiological, and neuro-cardiac axis remodeling to suppress ventricular arrhythmias. Methods Yorkshire pigs underwent sham surgery or anterior MI creation. Two weeks post-MI, animals were randomized to percutaneous epicardial resiniferatoxin (RTX, for cardiac-selective TRPV1 afferent depletion) or vehicle administration. Four weeks later, terminal studies assessed the effects of RTX on cardiac structure and function, ventricular arrhythmogenesis, and neuro-cardiac axis remodeling using in vivo electrophysiologic mapping, real-time neurotransmitter sensing, immunohistochemistry, and transcriptomic profiling. Results Cardiac TRPV1 afferent depletion was confirmed by blunted responses to TRPV1 agonists. RTX-treated animals exhibited improved left ventricular function, reduced end-diastolic diameter, and suppressed ventricular tachycardia/fibrillation (VT/VF) inducibility. Endocardial electroanatomic mapping revealed improved VT/VF electrophysiologic correlates in RTX-treated animals, including fewer deceleration zones and late potentials. Epicardial multielectrode mapping demonstrated reduced electrophysiologic heterogeneity in the scar border zone. Real-time release of adrenergic neurotransmitters (noradrenaline and neuropeptide Y) was normalized during sympathoexcitation in RTX-treated animals. Histologically, RTX treatment attenuated scar border zone myocardial fibrosis and sympathetic nerve sprouting, while suppressing T cell infiltration in cardiac sensory ganglia. Bulk RNA-sequencing of stellate ganglia revealed downregulation of adrenergic genes. Conclusion Cardiac TRPV1 afferent ablation post-completed MI alters disease trajectory by mitigating structural, functional, and neuro-cardiac axis remodeling. Targeting cardiac TRPV1 afferents represents a promising subacute post-MI therapeutic strategy.

physiology↗

bioRxiv2026

Nationwide multi-omics profiling of Japanese jack mackerel reveals geographic gut microbiome structuring despite host panmixia

Host genetic markers often fail to resolve regional origins in highly connected or panmictic marine species. The Japanese jack mackerel, Trachurus japonicus, is a commercially important fishery species around Japan that shows little or no detectable population structure. Here, we used nationwide multi-omics profiling to compare host genomic variation and gut microbiome composition in wild T. japonicus collected from coastal regions across Japan. We generated MIG-seq data for 43 individuals and 16S rRNA gene profiles for 24 individuals; after quality filtering, 19 individuals remained for matched host-microbiome comparison. Genome-wide host SNP analyses showed weak or absent geographic population structure, consistent with previous evidence of panmixia in Japanese waters. In contrast, gut microbiome composition showed geographic structuring based on Bray-Curtis dissimilarity and PERMANOVA, and this pattern was not explained by proximity to river mouths or host-related variables. Locality- or individual-associated bacterial lineages contributed to the observed differences in the microbiome, while chloroplast-associated and Cyanobacteria-assigned ASVs suggested recent dietary or environmental input. These results indicate that gut microbiome can show regional biological variation not apparent from host genetic markers alone. Our study provides a proof-of-concept example of integrating host genomics and gut microbiome profiling to evaluate regional characteristics and origins in highly connected marine animals.

microbiology↗

bioRxiv2026

High side chain promiscuity of the terminal enzyme in the homologation pathway for L-phenylalanine and L-tyrosine

Natural product (NPs) and their derivatives are a major source of small-molecule drugs, and the building blocks of these NPs are often amino acids. These include both proteinogenic and nonproteinogenic amino acids (NPAAs), the latter of which expand the structural diversity of NPs. Homologation, or the addition of a methylene group to the amino acid side chain, is one modification that generates NPAAs. If the natural homologation pathway can be characterized and engineered, it could be used to diversify NPs. In this study, we investigated the terminal enzyme of this pathway, HphB, to determine its substrate scope. HphB was tested with various substrates that differed in backbone and/or side chain structures relative to its natural substrate. The results showed that HphB exhibits high promiscuity toward substrates with different side chains while maintaining strict specificity for the substrate backbone. Comparative analysis with two homologous enzymes from primary metabolic pathways revealed that HphB displays markedly higher substrate promiscuity. Bioinformatics analysis and structural modeling suggest that this promiscuity arises from the absence of a "lid" over the active site, resulting in increased solvent exposure of the substrate side chain. This study highlights the unique substrate flexibility of HphB and is a step toward engineering the homologation pathway to generate amino acid derivatives.

biochemistry↗

bioRxiv2025

Amenability to Engineering of the Homologation Enzyme, HphA, through Homologous-Based Site-Directed Mutagenesis

Homologation of amino acids, the addition or deletion of a methylene group onto their side chains, has the potential to increase the biostability and bioavailability of peptide natural products. The first enzyme in the homologation pathway, HphA, has been previously characterized and is substrate selective. Bioinformatics studies were used to identify amino acids in the active site of HphA, which may play a role in substrate selection, by comparison to homologous enzymes, homocitrate synthase (HCS) and 2-isopropylmalate synthase (IPMS). Single point mutants to five amino acid residues in the HphAs active site were created to mimic those of HCS and IPMS. Their activities were measured via time-course assays with the natural substrates for HCS and IPMS. Residue A73 was identified as important in the substrate specificity of HphA; therefore, six different additional mutations were generated and tested with nine substrates with various side chains. The HphA A73L mutant exhibited the highest activity compared to the other mutants, showing activity with counterparts of L-Tyr (HphA natural substrate), L-Val (IPMS natural substrate), L-Leu, L-Ser, L-Trp, and L-Asp. Kinetic assays were taken with HphA A73L with the active substrates and compared with kinetic data from HphA WT, HCS, and IPMS. These results demonstrated that the A73L mutation significantly relaxed the substrate specificity of HphA, indicating its amenability to engineering. This research will serve as the foundation for future metabolic engineering studies on the enzymatic homologation pathway of amino acids.

bioengineering↗

bioRxiv2025

Autoantibodies targeting CRB2 and Nephrin correlate with extrafollicular B cells in idiopathic podocytopathies

Idiopathic podocytopathies lack mechanistic biomarkers and rely on empirical immunosuppression, yet the recent discovery of autoantibodies against slit diaphragm proteins implicates podocyte-directed autoimmunity as a driver of podocyte injury. However, the immune mechanisms underlying autoreactivity and phenotypic heterogeneity, particularly across age groups, remain unclear. Here, we identify expansion of CD21low T-bet+ atypical B cells (atBCs) in peripheral blood as a shared immunologic feature across idiopathic podocytopathies. In children, atBC accumulation was associated with heightened B cell activation, favoring extrafollicular responses and plasmablast differentiation. Serologic profiling revealed that autoantibodies against the slit diaphragm protein CRB2 are highly prevalent across disease phenotypes and frequently co-occur with anti-Nephrin antibodies, particularly in adults. Autoantibody abundance correlated with atBC expansion in affected children, implicating extrafollicular B cell responses as a key pathway for their generation, whereas adults showed additional involvement of follicular pathways. Complementary mouse modeling demonstrated intermolecular epitope spreading as a mechanism linking anti-CRB2 and anti-Nephrin autoreactivity. Together, these findings define a shared immune framework for idiopathic podocytopathies in which extrafollicular B cell responses drive autoreactivity to multiple slit diaphragm proteins, with implications for biomarker development and therapeutic stratification.

immunology↗

bioRxiv2025

MBD8 is required for LDL2-mediated transcriptional repression downstream of H3K9me2 in Arabidopsis

Histone H3 lysine 9 methylation (H3K9me) is a conserved epigenetic mark for transcriptional gene repression. In Arabidopsis, H3K9 dimethylation (H3K9me2) promotes the removal of a transcription-associated mark, H3K4 monomethylation (H3K4me1), and this process within genes requires the histone demethylase LDL2. However, how LDL2 specifically functions in genes that accumulate H3K9me2 remains unknown. Here, we show that methyl-CpG-binding domain protein 8 (MBD8) binds to LDL2 and plays a role in H3K4me1 removal and transcriptional gene repression downstream of H3K9me2. MBD8 preferentially binds GC-poor DNA, regardless of its cytosine methylation status. Interestingly, LDL2 protein levels were significantly reduced in the mbd8 mutant background, suggesting that MBD8 stabilizes the LDL2 protein, facilitating H3K4me1 removal and the transcriptional repression of H3K9me-marked genes. The domain necessary for the MBD8-LDL2 interaction is conserved among plants. We propose that MBD proteins have diverse functions beyond their methylated DNA-binding capabilities.

plant biology↗

bioRxiv2025

A Method for Predicting Enzyme Substrate Specificity Residues Using Homologous Sequence Information

Identifying amino acid residues that are critical for the catalytic function of enzymes is essential for elucidating reaction mechanisms, facilitating drug discovery, and advancing protein engineering. However, experimentally and computationally distinguishing residues that maintain structural integrity from those directly involved in enzymatic function remains a major challenge. In this study, we developed a methodology to identify amino acid residues that influence substrate specificity in enzymes with homologous structures. We framed the sequence comparison as a classification problem, treating each residue as a feature, thereby enabling the rapid and objective identification of key residues responsible for functional differences. To validate the proposed method, we applied it to three enzyme pairs-- trypsin/chymotrypsin, adenylyl cyclase/guanylyl cyclase, and lactate dehydrogenase (LDH)/malate dehydrogenase (MDH). The results confirmed the accurate prediction of previously identified specificity-determining residues. Furthermore, we conducted experiments on the LDH/MDH pair and successfully introduced mutations into key residues to alter substrate specificity, enabling LDH to utilize oxaloacetate while maintaining its expression levels. These findings demonstrate the potential of this method for efficiently identifying residues that govern substrate specificity. We have further developed this approach into a practical tool, the EZSCAN: Enzyme Substrate-specificity and Conservation Analysis Navigator (https://ezscan.pe-tools.com/), which enables rapid identification of amino acid residues critical for enzyme function. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/656053v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@d97b76org.highwire.dtl.DTLVardef@38bbe8org.highwire.dtl.DTLVardef@b8b6edorg.highwire.dtl.DTLVardef@f1b858_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

H3K4me2 orchestrates H2A.Z and Polycomb repressive marks in Arabidopsis

The dimethylation of histone H3 lysine 4 (H3K4me2) plays an important role in developmental phase transitions in plants, such as regeneration from the callus and the initiation of flowering. H3K4me2 in plants is correlated with transcriptionally repressed states, which can be accounted for by transcription-coupled active H3K4me2 demethylation, but the converse molecular pathway by which H3K4me2 represses transcription remains largely unexplored. Here, we show that H3K4me2 colocalizes with the facultatively repressive marks H2A.Z, H2A ubiquitination (H2Aub), and H3K27me3 in the gene body. Our genetic analyses reveal that H3K4me2 functions upstream but not downstream of these repressive marks. Interestingly, in genes with diurnal H3K4me2 oscillation, H3K4me2 oscillates in antiphase with transcription but in phase with H2A.Z. In addition, the genetic manipulation of H3K4me2 affects the oscillating profiles of H2A.Z, suggesting the efficient relay from H3K4me2 to H2A.Z. Notably, the diurnal oscillation of H2Aub is much weaker than that of H2A.Z despite the overall similarity in their distributions. These results suggest that H3K4me2 orchestrates H2A.Z and H2Aub with distinct dynamics. Our findings reveal that plants employ H3K4me2 to transmit transcriptional cessation into repressive chromatin states.

genetics↗

bioRxiv2025

Identification and functional characterization of toluene degradation genes in Acinetobacter sp. Tol 5

Microbial degradation of aromatic compounds provides sustainable solutions for environmental remediation and bioconversion. Acinetobacter sp. Tol 5 is notable for its strong adhesiveness and potential as a biocatalyst for toluene degradation; however, its toluene metabolic pathway has not been fully elucidated. In this study, genomic analysis identified a cluster of genes in Tol 5 highly similar to the well-known tod operon of Pseudomonas putida, encoding enzymes responsible for toluene metabolism. Phylogenetic analyses indicated that these tod genes, unusual among Acinetobacter species, were likely acquired through horizontal gene transfer. Transcriptomic analyses revealed that todF and todC1 are co-transcribed, while the adjacent fadL2 gene, encoding a putative outer membrane transporter corresponding to P. putida todX, is independently transcribed. Functional characterization using gene-knockout mutants revealed that TodC1, the large subunit of dioxygenase, is essential for growth on toluene, whereas FadL2 is not essential. Growth experiments further showed that the todC1 knockout mutant could metabolize benzoate, but not toluene or benzene, confirming that the TOD pathway is the primary route for toluene and benzene degradation in Tol 5. The identification of the functional TOD pathway, which is unique within Acinetobacter, provides genetic and biochemical insights for the development of Tol 5 as an efficient immobilized biocatalyst for the bioremediation and bioconversion of aromatic compounds.

microbiology↗

bioRxiv2024

A novel RyR2-selective stabilizer prevents and ameliorates severe arrhythmias in stress-induced ventricular tachycardia

Background and PurposeAberrant activation of the type 2 ryanodine receptor (RyR2) causes lethal arrhythmias, such as catecholaminergic polymorphic ventricular tachycardia (CPVT). Developing drugs that suppress RyR2 hyperactivation may be key to novel arrhythmia treatments. This study evaluated the antiarrhythmic potential of Ryanozole, a recently developed novel RyR2 modulator with high affinity and selectivity, using CPVT mouse models harboring mutant RyR2s. Experimental approachIn vitro effects of Ryanozole were evaluated by ER Ca2+-based assay and [3H]Ryanodine binding assay using RyR2-expressing HEK293 cells. Two lines of mice with different arrhythmia severity, RyR2-R420W and -K4750Q, were employed for in vivo assessments. Intracellular Ca2+ signals were analyzed in isolated cardiomyocytes using Cal520. Antiarrhythmic effects were evaluated by ECG under catecholaminergic challenge in anesthetized mice and during spontaneous arrhythmias in conscious mice. ECG and echocardiographic parameters were evaluated before and after drug administration. Key resultsRyanozole inhibited both wild type and mutant RyR2s with similar IC50 of 15-40 nM. The inhibition was more potent at lower cytosolic Ca2+ concentrations. It suppressed Ca2+ waves and Ca2+ sparks without affecting action potential-evoked Ca2+ transients. Ryanozole effectively prevented adrenaline-induced arrhythmias and rapidly terminated spontaneous arrhythmias during daily activity. Importantly, Ryanozole did not impair cardiac conduction or contractility, unlike conventional antiarrhythmic drugs. Conclusions and implicationsRyanozole strongly suppresses RyR2 under diastolic Ca2+ conditions, thereby preventing the arrhythmogenic trigger of aberrant Ca2+ release. This mechanism likely provides potent antiarrhythmic effects while preserving cardiac function. Ryanozole is a promising therapeutic candidate treating RyR2-mediated arrhythmias, such as CPVT.

pharmacology and toxicology↗

bioRxiv2024

The minimal SUF system can substitute for the canonical iron-sulfur cluster biosynthesis systems by using inorganic sulfide as the sulfur source.

Biosynthesis of iron-sulfur (Fe-S) clusters is indispensable for living cells. Three biosynthesis systems termed NIF, ISC and SUF have been extensively characterized in both bacteria and eukarya. For these L-cysteine is the sulfur source. A bioinformatic survey suggested the presence of a minimal SUF system composed of only two components, SufB* (a putative ancestral form of SufB and SufD) and SufC, in anaerobic archaea and bacteria. Here, we report the successful complementation of an Escherichia coli mutant devoid of the usual ISC and SUF systems upon expression of the archaeal sufB*C genes. Strikingly, this heterologous complementation occurred under anaerobic conditions only when sulfide was supplemented to the culture media. Mutational analysis and structural predictions suggest that the archaeal SufB*C most likely forms a SufB*2C2 complex and serves as the scaffold for de novo Fe-S cluster assembly using the essential Cys and Glu residues conserved between SufB* and SufB, in conjunction with a His residue shared between SufB* and SufD. We also demonstrate artificial conversion of the SufB*2C2 structure to the SufBC2D type by introducing several mutations to the two copies of sufB*. Our study thus elucidates the molecular function of this minimal SUF system and suggests that it is the evolutionary prototype of the canonical SUF system.

microbiology↗

bioRxiv2024

Intraoperative Ablation Control Based on Real-time Necrosis Monitoring Feedback: Numerical Evaluation

Ablation therapy is a type of minimally invasive treatment, utilized for various organs including the brain, heart, and kidneys. The accuracy of the ablation process is critically important to avoid both insufficient and excessive ablation, which may result in compromised efficacy or complications. The thermal ablation is formulated by two theoretical models: the heat transfer (HT) and necrosis formation (NF) models. In modern medical practices, feed-forward (FF) and temperature feedback (TFB) controls are primarily used as ablation control methodologies. FF involves pre-therapy procedure planning based on previous experiences and theoretical knowledge without monitoring the intraoperative tissue response, hence, it cant compensate for discrepancies in the assumed HT or NF models. These discrepancies can arise due to individual patients tissue characteristic differences and specific environmental conditions. Conversely, TFB control is based on the intraoperative temperature profile. It estimates the resulting heat damage based on the monitored temperature distribution and assumed NF model. Therefore, TFB can make necessary adjustments even if there is an error in the assumed HT model. TFB is thus seen as a more robust control method against modeling errors in the HT model. Still, TFB is limited as it assumes a fixed NF model, irrespective of the patient or the ablation technique used. An ideal solution to these limitations would be to actively monitor heat damage to the tissue during the operation and utilize this data to control ablation. This strategy is defined as necrosis feedback (NFB) in this study. Such real-time necrosis monitoring modalities making NFB possible are emerging, however, there is an absence of a generalized study that discusses the integration and quantifies the significance of the real-time necrosis monitor techniques for ablation therapy. Such an investigation is expected to clarify the universal principles of how these techniques would improve ablation therapy. In this study, we examine the potential of NFB in suppressing errors associated with the NF model as NFB is theoretically capable of monitoring and suppressing the errors associated with the NF models in its closed control loop. We simulate and compare the performances of TFB and NFB with artificially generated modeling errors using the finite element method (FEM). The results show that NFB provides more accurate ablation control than TFB when NF-oriented errors are applied, indicating NFBs potential to improve the ablation control accuracy and highlighting the value of the ongoing research to make real-time necrosis monitoring a clinically viable option.

bioengineering↗

bioRxiv2023

FGF9, a potent mitogen, is a new ligand for integrin αvβ3, and the FGF9 mutant defective in integrin binding acts as an antagonist.

FGF9 is a potent mitogen and survival factor, but FGF9 protein level is generally low and restricted to a few adult organs. Aberrant expression of FGF9 usually results in cancer. However, the mechanism of FGF9 action has not been fully established. Previous studies showed that FGF1 and FGF2 directly bind to integrin v{beta}3 and this interaction is critical for signaling functions (FGF-integrin crosstalk). FGF1 and FGF2 mutants defective in integrin binding were defective in signaling, whereas the mutants still bound to FGFR, and suppressed angiogenesis and tumor growth, indicating that they act as antagonists. We hypothesize that FGF9 requires direct integrin binding for signaling. Here we show that docking simulation of interaction between FGF9 and v{beta}3 predicted that FGF9 binds to the classical ligand-binding site of v{beta}3. We showed that FGF9 actually bound to integrin v{beta}3, and generated an FGF9 mutants in the predicted integrin-binding interface. An FGF9 mutant (R108E) was defective in integrin binding, activating FRS2 and ERK1/2, inducing DNA synthesis, cancer cell migration, and invasion in vitro. R108E suppressed DNA synthesis induced by WT FGF9 and suppressed DNA synthesis and activation of FRS2 and ERK1/2 induced by WT FGF9 (dominant-negative effect). These findings indicate that FGF9 requires direct integrin binding for signaling and that R108E has potential as an antagonist to FGF9 signaling.

biochemistry↗

bioRxiv2023

Precise immunofluorescence canceling for highly multiplexed imaging capturing specific cell state

Cell states are regulated by the response of signaling pathways to receptor ligand-binding and inter-cellular interactions. High-resolution imaging has been attempted to explore the dynamics of these processes and, recently, multiplexed imaging has profiled cell states by achieving a comprehensive acquisition of spatial protein information from cells. However, the specificity of antibodies is still compromised when visualizing activated signals. Here, we developed Precise Emission Canceling Antibodies (PECAbs) that have cleavable fluorescent labeling. PECAbs enable high-specificity sequential imaging using hundreds of antibodies, allowing for reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system can serve as a comprehensive platform for analyzing complex cell processes.

cell biology↗

bioRxiv2023

Screening for novel RyR2 inhibitors by ER Ca2+ monitoring

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel on the endoplasmic/sarcoplasmic reticulum (ER/SR) that plays a central role in the excitation-contraction coupling in the heart. Hyperactivity of RyR2 has been linked to ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT) and heart failure, where spontaneous Ca2+ release via hyperactivated RyR2 depolarizes diastolic membrane potential to induce triggered activity. In such cases, drugs that suppress RyR2 activity are expected to prevent the arrhythmias, but there is no clinically available RyR2 inhibitors at present. In this study, we searched for RyR2 inhibitors from a well-characterized compound library using a recently developed ER Ca2+-based assay, where the inhibition of RyR2 activity was detected by the increase in ER Ca2+ signals from R-CEPIA1er, a genetically encoded ER Ca2+ indicator, in RyR2-expressing HEK293 cells. By screening 1535 compounds in the library, we identified three compounds (chloroxylenol, methyl orsellinate and riluzole) that greatly increased the ER Ca2+ signal. All of the three compounds suppressed spontaneous Ca2+ oscillations in RyR2-expressing HEK293 cells and correspondingly reduced the Ca2+-dependent [3H]ryanodine binding activity. In cardiomyocytes from RyR2-mutant mice, the three compounds effectively suppressed abnormal Ca2+ waves without substantial effects on the action-potential-induced Ca2+ transients. These results confirm that ER Ca2+-based screening is useful for identifying modulators of ER Ca2+ release channels and suggest that RyR2 inhibitors have potential to be developed as a new category of antiarrhythmic drugs. Significance statementWe successfully identified three compounds having RyR2 inhibitory action from a well-characterized compound library using an ER Ca2+-based assay, and demonstrated that these compounds suppressed arrhythmogenic Ca2+ wave generation without substantially affecting physiological action-potential induced Ca2+ transients in cardiomyocytes. This study will facilitate the development of RyR2 specific inhibitors as a potential new class of drugs for life-threatening arrhythmias induced by hyperactivation of RyR2.

pharmacology and toxicology↗

bioRxiv2023

Cell polarity linked to gravity sensing is generated by protein translocation from statoliths to the plasma membrane.

Organisms have evolved under the gravitational force and sense the direction of gravity via statoliths in specialized cells. In the gravitropism of flowering plants, the starch-accumulating plastids, amyloplasts, in gravity sensing cells act as statoliths. The gravity sensing mechanism has long been considered a mechanosensing process by which amyloplasts transmit forces to intracellular structures, but the molecular support has not been reported. This study revealed that LAZY1-LIKE family proteins involved in gravity signaling in statocytes are localized to the amyloplast periphery and its proximal plasma membrane, resulting in polar localization according to the direction of gravity. We propose a gravity sensing mechanism by which LZY transmits the positional information of amyloplasts, i.e., the direction of gravity, by translocating to the plasma membrane.

plant biology↗

bioRxiv2023

Cotranscriptional demethylation induces global loss of H3K4me2 from active genes in Arabidopsis

Based on studies of animals and yeasts, methylation of histone H3 lysine 4 (H3K4me1/2/3, for mono-, di-, and tri-methylation, respectively) is regarded as the key epigenetic modification of transcriptionally active genes. In plants, however, H3K4me2 correlates negatively with transcription, and the regulatory mechanisms of this counterintuitive H3K4me2 distribution in plants remain largely unexplored. A previous genetic screen for factors regulating plant regeneration identified Arabidopsis LYSINE-SPECIFIC DEMETHYLASE 1-LIKE 3 (LDL3), which is a major H3K4me2 demethylase. Here, we show that LDL3-mediated H3K4me2 demethylation depends on the transcription elongation factor Paf1C and phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII). In addition, LDL3 binds to phosphorylated RNAPII. These results suggest that LDL3 is recruited to transcribed genes by binding to elongating RNAPII and demethylates H3K4me2 cotranscriptionally. Importantly, the negative correlation between H3K4me2 and transcription is disrupted in the ldl3 mutant, demonstrating the genome-wide impacts of the transcription-driven LDL3 pathway to control H3K4me2 in plants. Our findings implicate H3K4me2 in plants as chromatin memory for transcriptionally repressive states, which ensures robust gene control.

plant biology↗