Biology subjects

Nagai, A.

Publications and source records attributed to Nagai, A..

5 recordsLinked to original sources

bioRxiv2026

THE STRUCTURES OF MATURE AND IMMATURE ST. LOUIS ENCEPHALITIS VIRUS REVEAL CONSERVED HISTIDINE RESIDUES AFFECTING VIRUS FITNESS

Orthoflaviviruses undergo significant structural changes through maturation and infection, yet the molecular mechanisms remain incompletely understood. Using St. Louis encephalitis virus (SLEV) as a model, a reemerging mosquito-borne orthoflavivirus endemic in the Americas, we elucidated the structures of immature and mature SLEV particles at resolutions of 4.4 [A] and 3.3 [A], respectively, using cryo-EM. SLEV is characterized by glycosylated E and prM proteins, the presence of lipid pockets, and is stabilized by an intricate network of inter- and intra-protein interactions between E and (pr)M across maturation stages. Several interactions were mediated by histidines that play different roles depending on SLEV maturation and pH. Non-lethal single mutations of H285R and H443R in E protein affect SLEV replication in mammalian and mosquito cell lines, and delay death in mouse models of infection. These histidine residues are conserved across orthoflaviviruses, illustrating the complexity of orthoflavivirus particles and indicating a possible strategy for attenuation.

microbiology↗

bioRxiv2026

Regulation of sphingolipid synthesis by the C2H2 zinc finger transcription factor Com2 through ubiquitin-proteasome mediated degradation pathway

Membrane lipid synthesis is globally coordinated by a limited set of master transcription factors that regulate broad gene networks encoding lipid-metabolic enzymes and their regulators. Here, we identify the C2H2 zinc-finger transcription factor Com2 as a regulator of sphingolipid homeostasis in Saccharomyces cerevisiae that promotes transcription of downstream targets, including the protein kinase Ypk1, a key activator of sphingolipid synthesis. Com2 protein abundance increased upon treatment with myriocin, an inhibitor of sphingolipid synthesis, but rapidly decreased after addition of phytosphingosine (PHS), a precursor of complex sphingolipids; this decrease was blocked by proteasome inhibitors. These results suggest that Com2 is regulated in a sphingolipid-dependent manner through proteasome-mediated degradation. Moreover, a Com2 mutant in which lysine residues putatively involved in ubiquitination were replaced with arginine exhibited attenuated PHS-dependent degradation and elevated phosphorylation. Likewise, a mutant in which putative phosphorylation sites were replaced with alanine showed reduced PHS-dependent degradation. Together, these findings indicate that Com2 undergoes phosphorylation-dependent degradation via the ubiquitin-proteasome system in response to sphingolipid levels.

cell biology↗

bioRxiv2025

ALKBH1 drives codon-biased, pro-oncogenic translation and tumor microenvironment remodeling in glioma via tRNA wobble oxidation

Gliomas rely on translational plasticity to sustain heterogeneity, stemness, and immune evasion. Here we identify the tRNA dioxygenase ALKBH1 as a central regulator of codon-biased translation and tumor microenvironment remodeling. LC-MS/MS profiling of patient tumors revealed enrichment of ALKBH1-mediated wobble cytidine oxidation modifications in high-grade gliomas. Genetic perturbation demonstrated that ALKBH1 overexpression slows proliferation in vitro but worsens survival in vivo by promoting glioma stem-like and neuronal states and suppressing anti-tumor immunity. Ribosome profiling showed that ALKBH1 establishes an A/T-ending codon-biased translational program, enhancing decoding of rare leucine codons (TTA/TTG) and driving synthesis of pro-stemness transcripts. Single-cell RNA-seq analyses further revealed that ALKBH1 promotes glioma heterogeneity and induces neuronal cell clusters and rewires intercellular communication ECM-related signaling while dampening immune pathways and reducing immune infiltration. These findings establish a mechanistic link between tRNA oxidation, codon bias, and glioma aggressiveness, positioning ALKBH1 as a potential therapeutic target for glioma.

cancer biology↗

bioRxiv2025

Influence of Substrate Elastic Modulus on Cell Shape and Displacement During the Motility Cycle

Cardiac fibroblasts play a pivotal role in heart tissue dynamics, responding to mechanical cues from their environment. This study examines how substrate elasticity influences the motility and shape of chicken embryonic cardiac fibroblasts on polydimethylsiloxane (PDMS) substrates of different stiffness (10 kPa and 400 kPa). Using time-lapse phase-contrast microscopy, we identified distinct motility cycles characterized by elongation, contraction, and retraction phases. On soft substrates, cells exhibited slower retraction speeds and longer motility cycles compared to those on hard substrates. Notably, cells on soft substrates left trailing edges upon retraction, leading to pronounced morphological oscillations, whereas cells on hard substrates adopted near-circular shapes with less coordinated motility. Quantitative analyses revealed that the duration of the static phase was significantly shorter on hard substrates, correlating with differences in cell shape parameters, including length, circularity, and area. These findings suggest that substrate elasticity modulates the balance between cell-substrate adhesion and contractile forces, influencing cell polarity and motility. Our work advances the understanding of how mechanical environments shape fibroblast dynamics, offering insights relevant to tissue engineering and fibrosis research. SIGNIFICANCEMechanical cues from the cellular microenvironment are known to regulate cell behavior, yet how substrate stiffness shapes the dynamic motility cycle of fibroblasts remains incompletely understood. Here, we use high-resolution time-lapse imaging to reveal that cardiac fibroblasts on soft (10 kPa) PDMS undergo polarized, oscillatory migration with prolonged static phases, while those on stiff (400 kPa) substrates exhibit rapid retraction and random-like displacement with reduced polarity. These findings highlight how substrate elasticity modulates the interplay between traction forces and adhesion stability, governing directionality and morphology. This work provides mechanobiological insights into fibroblast behavior with implications for designing biomaterials to guide tissue remodeling and fibrosis progression.

biophysics↗

bioRxiv2020

OTTER, a new method quantifying absolute amounts of tRNAs

To maintain optimal proteome, both codon choice of each mRNA and supply of aminoacyl-tRNAs are two principal factors in translation. Recent reports revealed that tRNAs are more dynamic in amount than we had expected. High-throughput methods such as RNA-Seq or microarray are versatile for comprehensive analyses of changes in individual tRNA amounts, but they suffer from lack of assessment of signal production efficiency of individual tRNA species. Thus, they are not the perfect choice to measure absolute amounts of tRNA. Here, we introduce a novel method for this purpose, oligonucleotide-directed three-primer terminal extension of RNA (OTTER), which employs fluorescence labeling at the 3-terminus of a specific tRNA by optimized reverse primer extension and an assessment step of each labeling efficiency by Northern blotting. We quantified absolute amounts of 34 individual and 4 pairs of isoacceptor tRNAs out of the total 42 nuclear-encoded isoacceptors in the yeast Saccharomyces cerevisiae. We revealed that the amounts of tRNAs are in the range of 0.030-0.73 pmol/g RNA in the yeast cells logarithmically grown in a rich glucose medium. The tRNA amounts seem to be regulated at the isoacceptor level by a few folds according to physiological growing conditions. Data obtained by OTTER are poorly correlated with those by simple RNA-Seq and only marginally with those by microarray. However, the OTTER data are good agreement with those by 2D-gel analysis of in vivo radiolabeled RNA samples. Thus, OTTER is a suitable method for quantifying absolute amounts of tRNAs in the isoacceptor resolution.

molecular biology↗