Biology subjects

Furukawa, H.

Publications and source records attributed to Furukawa, H..

10 recordsLinked to original sources

bioRxiv2026

Systematic analysis of intracellular replication competence of mammalian, avian, and reptilian kolmioviruses.

Kolmioviruses (KoVs) are circular, single-stranded RNA viruses whose evolution may have involved host switching between vertebrate classes. However, the host specificity of their intracellular replication remains poorly understood. Here, we examined six mammalian, one avian, and one reptilian KoVs using cell lines derived from the same three vertebrate classes. We transfected cells with plasmids encoding 1.2x genome-length KoV sequences and assessed persistent replication by monitoring delta antigen-positive cells for up to 18 days. Four mammalian KoVs replicated persistently only in mammalian cell lines, whereas avian and reptilian KoVs replicated persistently only in both avian and reptilian cell lines. By contrast, the remaining two mammalian KoVs, nested within the clade whose deepest lineages are the avian and reptilian KoVs, replicated in cell lines from all three vertebrate classes. The observed patterns of persistent replication are concordant with the phylogenetic grouping of the KoVs. Our findings support a possible evolutionary scenario in which the common ancestor of the two mammalian KoVs in the avian-reptilian clade acquired compatibility with mammalian cells while retaining the capacity to replicate in avian and reptilian cells, contributing to the transition of this lineage to mammalian hosts.

microbiology↗

bioRxiv2026

Artificial initiation codons and engineered initiator tRNAs enable N-terminal noncanonical amino acid incorporation in intact cell-free translation systems

Noncanonical amino acid (ncAA) incorporation at the protein N-terminus provides a powerful strategy for installing defined chemical handles while minimizing perturbation of internal protein sequences. However, highly efficient initiation-based ncAA incorporation systems suppress the native methionine pathway by removing methionine or methionyl-tRNA synthetase, limiting their use for proteins containing internal methionine residues. Here, we developed an orthogonal initiation system for selective N-terminal ncAA incorporation into proteins in intact cell-free translation systems. We systematically profiled background initiation from all 64 codons in reconstituted translation systems and identified low-background artificial initiation codons. Engineered initiator tRNAs, termed tRNAIniTx, were then designed to decode selected codons and support ncAA-dependent initiation. The optimized CAC/tRNAIniTx04GUG pair enabled efficient N-terminal incorporation of N-biotinyl-L-phenylalanine without removing methionine or methionyl-tRNA synthetase, reaching over 90% incorporation. The system was further extended to p-azido-L-phenylalanine and to an Escherichia coli extract-based cell-free translation system. Finally, N-terminally biotinylated proteins were directly immobilized on streptavidin biosensors for purification-free biolayer interferometry analysis of computationally designed Brd4BD2 binders. This work establishes a codon-guided orthogonal initiation strategy for N-terminal protein functionalization while preserving the native methionine translation pathway.

synthetic biology↗

bioRxiv2025

On the mechanism of calcium permeability and magnesium block in NMDA receptors - a central molecular paradigm in neuroplasticity

Neuroplasticity is a fundamental cellular mechanism underlying learning and memory formation and is primed by the coincidental detection of neurotransmitter release from the presynapse and the subsequent calcium influx upon voltage change in the postsynaptic membrane (Bliss and Collingridge, 1993). Molecular assemblies that achieve these events are N-methyl-D-aspartate receptors (NMDARs), which bind the neurotransmitter glutamate and a co-agonist, either glycine or D-serine, and allow Ca2+ influx upon relief of the Mg2+ channel blockade by membrane depolarization. However, the molecular basis governing Ca2+ permeability and Mg2+ blockade in NMDAR remains limited. Here, we demonstrate that Ca{superscript 2} permeation through the narrow constriction of the cation selectivity filter involves partial dehydration, as evidenced by multiple Ca{superscript 2} binding sites captured using single-particle cryo-electron microscopy (cryo-EM). In contrast, Mg2+ binds outside of the selectivity filter through the water network by remaining hydrated, thereby serving as a channel blocker. Furthermore, we show that the lipid network around the selectivity filter influences the stability of Mg2+ binding. Our study details the critical transmembrane chemistry of NMDAR for initiating neuroplasticity.

neuroscience↗

bioRxiv2025

Non-catalytic and catalytic TREHALOSE-6-PHOSPHATE SYNTHASES interact with RAMOSA3 to control maize development.

Trehalose-6-phosphate (Tre6P) is the intermediate in the two-step pathway of trehalose biosynthesis mediated by Tre6P-synthases (TPSs) and Tre6P-phosphatases (TPPs). Plants harbor small families of TPS and TPP genes, however most plant TPSs lack enzymatic activity, suggesting they have regulatory functions. The classical mutant ramosa3 (ra3) increases inflorescence branching in maize, and RA3 encodes a catalytic TPP. We found that RA3 interacts with maize ZmTPS1, a non-catalytic TPS. Mutants in ZmTPS1 and its close paralog ZmTPS12 enhance ra3 phenotypes, suggesting their physical interaction is biologically significant. ZmTPS1 also interacts with the two catalytically active maize TPSs, ZmTPS11 and ZmTPS14, however zmtps11;zmtps14 double mutants fail to complete embryogenesis, suggesting that they are essential, as in arabidopsis. Interestingly, the non-catalytic ZmTPS1 protein stimulated the coupled activity of RA3 and ZmTPS14, suggesting that RA3, ZmTPS1, and ZmTPS14 form a complex, and we confirmed this by expressing and purifying the three proteins and by Alphafold predictions. Our results suggest that non-catalytic TPSs form a complex with catalytic TPSs and TPPs to stimulate catalytic activity and regulate plant development.

plant biology↗

bioRxiv2024

It only takes seconds for a human monoclonal autoantibody to inhibit N-methyl-D-aspartate receptors

Transfer of autoantibodies targeting ionotropic N-methyl-D-aspartate receptors in autoimmune encephalitis patients into mice leads to typical disease signs. Long-term effects of the pathogenic antibodies consist of immunoglobulin G-induced crosslinking and receptor internalization. We focused on the direct and immediate impact of a specific pathogenic patient-derived monoclonal autoantibody (immunoglobulin G #003-102) on receptor function. We performed cell-attached recordings in cells transfected with the GluN1 and GluN2A subunit of the N-methyl-D-aspartate receptor. Immunoglobulin G #003-102 binds to the amino-terminal domain of the glycine-binding GluN1 subunit. It reduced simultaneous receptor openings significantly compared to controls at both low and high glutamate and glycine concentrations. Closer examination of our data in 50-second to 2-second intervals revealed, that Immunoglobulin G #003-102 rapidly decreases the number of open receptors. However, antigen-binding fragments of immunoglobulin G #003-102 did not reduce the receptor openings. In conclusion, patient-derived immunoglobulin G #003-102 inhibits N-methyl-D-aspartate receptors rapidly and directly before receptor internalization occurs and the entire immunoglobulin G is necessary for this acute inhibitory effect. This suggests an application of the antigen-binding fragment-like constructs of #003-102 as a potential new treatment strategy for shielding the pathogenic epitopes on the N-methyl-D-aspartate receptors.

neuroscience↗

bioRxiv2024

Bayesian estimation of trunk-leg coordination during walking using phase oscillator models

In human walking, the legs and other body parts coordinate to produce a rhythm with appropriate phase relationships. From the point of view for rehabilitating gait disorders, such as Parkinson Disorders, it is important to understand the control mechanism of the gait rhythm. A previous study showed that the antiphase relationship of the two legs during walking is not strictly controlled using the reduction of the motion of the legs during walking to coupled phase oscillators. However, the control mechanisms other than those of the legs remains unknown. In particular, the trunk moves in tandem with the legs and must play an important role in stabilizing walking because it is above the legs and accounts for more than half of the mass of the human body. This study aims to uncover the control mechanism of the leg-trunk coordination in the sagittal plane using the coupled phase oscillators model and Bayesian estimation. We demonstrate that the leg-trunk coordination is not strictly controlled, as well as the interleg coordination.

bioengineering↗

bioRxiv2023

Dendritic, delayed, and stochastic CaMKII activation underlies behavioral time scale plasticity in CA1 synapses

Behavioral time scale plasticity (BTSP), is a form of non-Hebbian plasticity induced by integrating pre- and postsynaptic components separated by behavioral time scale (seconds). BTSP in the hippocampal CA1 neurons underlies place cell formation. However, the molecular mechanisms underlying this behavioral time scale (eligibility trace) and synapse specificity are unknown. CaMKII can be activated in a synapse-specific manner and remain active for a few seconds, making it a compelling candidate for the eligibility trace during BTSP. Here, we show that BTSP can be induced in a single dendritic spine using 2-photon glutamate uncaging paired with postsynaptic current injection temporally separated by behavioral time scale. Using an improved CaMKII sensor, we saw no detectable CaMKII activation during this BTSP induction. Instead, we observed a dendritic, delayed, and stochastic CaMKII activation (DDSC) associated with Ca2+ influx and plateau 20-40 s after BTSP induction. DDSC requires both pre-and postsynaptic activity, suggesting that CaMKII can integrate these two signals. Also, optogenetically blocking CaMKII 30 s after the BTSP protocol inhibited synaptic potentiation, indicating that DDSC is an essential mechanism of BTSP. IP3-dependent intracellular Ca2+ release facilitates both DDSC and BTSP. Thus, our study suggests that the non-synapse specific CaMKII activation provides an instructive signal with an extensive time window over tens of seconds during BTSP.

neuroscience↗

bioRxiv2022

Excitatory and inhibitory D-serine binding to the NMDA receptor

N-methyl-D-aspartate receptors (NMDARs) uniquely require binding of two different neurotransmitter agonists for synaptic transmission. D-serine and glycine bind to one subunit, GluN1, while glutamate binds to the other, GluN2. These agonists bind to the receptors bi-lobed ligand-binding domains (LBDs), which close around the agonist during receptor activation. To better understand the unexplored mechanisms by which D-serine contributes to receptor activation, we performed multi-microsecond molecular dynamics simulations of the GluN1/GluN2A LBD dimer with free D-serine and glutamate agonists. Surprisingly, we observed D-serine binding to both GluN1 and GluN2A LBDs, suggesting that D-serine competes with glutamate for binding to GluN2A. This mechanism is confirmed by our electrophysiology experiments, which show that D-serine is indeed inhibitory at high concentrations. Although free energy calculations indicate that D-serine stabilizes the closed GluN2A LBD, its inhibitory behavior suggests that it either does not remain bound long enough or does not generate sufficient force for ion channel gating. We developed a workflow using pathway similarity analysis to identify groups of residues working together to promote binding. These conformation-dependent pathways were not significantly impacted by the presence of N-linked glycans, which act primarily by interacting with the LBD bottom lobe to stabilize the closed LBD.

biophysics↗

bioRxiv2019

The Cryo-EM Structure of a Pannexin 1 Channel Reveals an Extracellular Gating Mechanism

Pannexins are large-pore forming channels responsible for ATP release under a variety of physiological and pathological conditions. Although predicted to share similar membrane topology with other large-pore forming proteins such as connexins, innexins, and LRRC8, pannexins have minimal sequence similarity to these protein families. Here, we present the cryo-EM structure of a pannexin 1 (Panx1) channel at 3.0 [A]. We find that Panx1 protomers harbor four transmembrane helices similar in arrangement to other large-pore forming proteins but assemble as a heptameric channel with a unique gate formed by Trp74 in the extracellular loop. Mutating Trp74 or the nearby Arg75 disrupt ion selectivity whereas altering residues in the hydrophobic groove formed by the two extracellular loops abrogates channel inhibition by carbenoxolone. Our structural and functional study establishes the extracellular loops as the unique structural determinants for channel gating and inhibition in Panx1 thereby providing the founding model to study pannexins.

neuroscience↗

bioRxiv2019

Structure and assembly of calcium homeostasis modulator proteins

Biological membranes of many tissues and organs contain large-pore channels designed to permeate a wide variety of ions and metabolites. Examples include connexin, innexin, and pannexin, which form gap junctions and/or bona fide cell surface channels. The most recently identified large-pore channels are the calcium homeostasis modulators (CALHMs), which permeate ions and ATP in a voltage-dependent manner to control neuronal excitability, taste signaling, and pathologies of depression and Alzheimers disease. Despite such critical biological roles, the structures and patterns of oligomeric assembly remain unclear. Here, we reveal the first structures of two CALHMs, CALHM1 and CALHM2, by single particle cryo-electron microscopy, which show novel assembly of the four transmembrane helices into channels of 8-mers and 11-mers, respectively. Furthermore, molecular dynamics simulations suggest that lipids can favorably assemble into a bilayer within the larger CALHM2 pore, but not within CALHM1, demonstrating the potential correlation between pore-size, lipid accommodation, and channel activity.

biophysics↗