Biology subjects

Yoshimi, A.

Publications and source records attributed to Yoshimi, A..

14 recordsLinked to original sources

bioRxiv2026

NECTIN4 defines a subtype-dependent tumor state coupling enfortumab vedotin targetability to antitumor immunity in bladder cancer

NECTIN4 is the target of enfortumab vedotin (EV), but its biological role in bladder cancer (BC) and its relationship to EV plus PD-1 blockade remain unclear. Integrating clinical multi-omics, spatial and single-cell immune profiling, and experimental models, we identify NECTIN4 as a subtype-dependent determinant of tumor state and antitumor immunity. In basal BC cells, NECTIN4 suppressed inflammatory and mesenchymal programs. Consistently, NECTIN4-high basal/squamous muscle-invasive BCs showed favorable survival, higher checkpoint inhibitor response rates, and a coordinated CD8+ T cell-inflamed microenvironment with no comparable associations in luminal papillary tumors. In orthotopic models, murine Nectin4 promoted CD8+ T cell-dependent tumor control, whereas human NECTIN4 conferred EV sensitivity. EV also attenuated NECTIN4-dependent TIGIT binding. Conversely, NECTIN4-low models were preferentially sensitive to HDAC inhibition. NECTIN4 therefore couples antibody-drug conjugate targetability to a subtype-specific tumor-immune state permissive for checkpoint blockade, whereas the NECTIN4-low state defines a distinct inflammatory-mesenchymal state with an alternative therapeutic vulnerability.

cancer biology↗

bioRxiv2026

N-terminal intrinsically disordered region mediates self-catalytic interfacial nucleation of Aspergillus oryzae hydrophobin RolA

Hydrophobins are biosurfactant proteins that coat the cell surfaces of filamentous fungi. On the conidial surface, hydrophobins self-assemble into rodlets, forming a dense hydrophobic film that promotes air-dispersibility. Although rodlet formation is closely associated with the physiology of filamentous fungi, its underlying molecular mechanisms remain largely unknown. Previously, we revealed that RolA, a hydrophobin derived from Aspergillus oryzae, forms rodlets at the air-water interface. In this study, we focused on the flexible N-terminal region of RolA, which lacks a well-defined tertiary structure, and hypothesized that this intrinsically disordered region regulates rodlet formation. To investigate its role, we used RolA mutants with reduced charges in the N-terminal region and analyzed the rodlet formation process on the surface of a water-in-air sessile droplet using atomic force microscopy. In addition, we quantitatively characterized rodlet formation at the air-water interface by applying a kinetic perspective to the interfacial tension change profiles obtained from dynamic surface tension measurements. The results suggested that RolA first forms a monolayer at the air-water interface, then rodlet formation proceeds through the continuous supply of free RolA monomers from the bulk phase to the interfacial RolA film. Our molecular dynamics simulations of RolA at the interface supported a model in which RolA molecules within the interfacial film interact with free monomers in the bulk phase through their N-terminal regions. These results reveal a previously unidentified role of the N-terminal region in rodlet formation and provide a more comprehensive framework for understanding the molecular mechanism underlying RolA rodlet formation.

biophysics↗

bioRxiv2026

Improved recombinant protein production and scale-up fermentation of Aspergillus oryzae hyphal-dispersion hydrophobin-deficient strain

In liquid fermentation of filamentous fungi such as Aspergillus oryzae, increased broth viscosity and biomass adhesion to bioreactor surfaces remain major challenges. We previously developed a hyphal dispersion mutant lacking two hyphal adhesion factors, namely cell wall -1,3-glucan (AG) and biofilm galactosaminogalactan (GAG) (AG{Delta}-GAG{Delta} strain). The culture broth of the AG{Delta}-GAG{Delta} strain has low viscosity, which improves mixing and enzyme production. However, mycelia still extensively attach to bioreactor walls and downstream equipment, which impairs mixing and reduces product recovery. The hydrophobin RolA, a surface-active protein of A. oryzae, densely coats conidia and hyphae and contributes to cell surface hydrophobicity. In this study, we disrupted the rolA gene in AG{Delta}-GAG{Delta} (AG{Delta}-GAG{Delta}-{Delta}rolA strain) and evaluated the effects of this disruption on hyphal adhesion to the walls of culture vessels, enzyme production, and bioreactor performance. At the flask scale, the adhesion to glass surfaces was significantly reduced and recombinant enzyme activity was increased by 10%. Improved culture recovery at the end of fermentation further increased total enzyme yield. In a lab-scale stirred-tank bioreactor, both growth and enzyme production were increased. Scaling-up to a 200-L bioreactor showed reduced agitation power consumption while improving hydrodynamic properties. Fermentation of AG{Delta}-GAG{Delta}-{Delta}rolA was successfully scaled up to a 3000-L bioreactor; consistent enzyme activity and improved flow circulation in the bioreactors were confirmed by computational fluid dynamics analysis. Overall, the AG{Delta}-GAG{Delta}-{Delta}rolA strain has increased enzyme production and scalability, supporting its suitability for industrial applications.

bioengineering↗

bioRxiv2026

A positional and combinatorial regulatory code for alternative splicing

Alternative pre-mRNA splicing generates extensive transcript diversity, yet the regulatory code that determines how splicing decisions are encoded across the transcriptome remains poorly defined. Splicing outcomes are controlled by combinatorial RNA-binding protein (RBP) interactions and positional context, but how these features are integrated at the transcriptome scale remains unclear. CLIP-based approaches have mapped RBP binding, but directly comparable endogenous maps across multiple RBPs are lacking, limiting inference of global regulatory principles. Here we introduce SCALE-CLIP, an endogenous CLIP framework that integrates CRISPR-Cas9-mediated epitope tagging with long-read-guided read attribution to generate directly comparable RBP binding maps across splicing-regulatory factors. Applied to 23 RBPs, SCALE-CLIP expanded endogenous RBP coverage and, across benchmarked shared factors, increased peak recovery by a median of 12.2-fold relative to ENCODE eCLIP while preserving specificity and reproducibility. We define a transcriptome-wide positional and combinatorial code for alternative splicing, in which binding position is a primary determinant of regulatory outcome: SRSF binding within alternative exons promotes inclusion, whereas binding on flanking exons drives exon skipping. Higher-order SRSF occupancy further tunes this code, buffering exon inclusion when centered on alternative exons but reinforcing repression when distributed across flanking exons. We also show that m6A provides an epitranscriptomic layer that locally enhances SRSF binding and is associated with increased exon inclusion. Together, these results establish a multi-layered RNA-binding logic in which binding position, combinatorial RBP architecture and RNA modification jointly shape splicing outcomes, providing a framework for rational interpretation and modulation of alternative splicing.

molecular biology↗

bioRxiv2026

Amorphous-to-Rodlet Structural Transition Governs the Interfacial Functions of Aspergillus oryzae Hydrophobin RolA

Hydrophobins are low-molecular-weight biosurfactant proteins that coat the cell surface of filamentous fungi, making it hydrophobic and supporting morphogenesis. On conidia, hydrophobins self-assemble to form rod-shaped multimeric structures known as rodlets. Previously, we reported that hydrophobin RolA from the industrial fungus Aspergillus oryzae first forms an amorphous film at the air-water interface and then undergoes structural rearrangement to form a densely packed rodlet film. This raised the question of whether the amorphous or the rodlet film is more important for the biological functions of RolA. In this study, to compare the properties of amorphous films with those of rodlet films, we used RolA mutants that had lost the ability to form rodlets and therefore remained in the amorphous state. We found that the rodlet film was more rigid than the amorphous film and had stronger surface activity and a greater capacity to change surface wettability. RolA altered the properties of A. oryzae conidia only when it was in the rodlet state. These findings highlight the functional versatility of RolA and show that their dynamic structural transitions directly modulate their function.

microbiology↗

bioRxiv2025

High-speed atomic force microscopy reveals surface-catalyzed elongation mechanism of fungal functional amyloid, hydrophobin RolA

Hydrophobins, a type of functional amyloid, are conserved in filamentous fungi and act as a protective coat in the fibrous form called rodlet. Rodlets form hierarchical structures where they are bundled and densely aligned, contributing to the hydrophobicity of the mycelium surface. However, the formation mechanism of the hierarchical structures is completely unknown. In this study we used high-speed atomic force microscopy to directly observe the structural dynamics of hierarchical structure formation by hydrophobin RolA from the industrial fungus Aspergillus oryzae at a single-fibril level and revealed its mechanism. The elongation of rodlets occurred at both ends and was discontinuous, alternating between periods when they could elongate (growth state) and could not elongate (pause state). This suggests an equilibrium of two distinct structural states at the rodlet ends. We also discovered an aggregation pathway, termed "surface-catalyzed elongation", in which elongation is promoted by lateral interactions between bundled rodlets. Surface-catalyzed elongation decreased the energy barrier of both structural switching between growth and pause states and elongation at rodlet ends, doubling the elongation rate in bundled rodlets. The rodlet surface could be considered as a catalyst for the elongation of neighboring rodlets. Surface-catalyzed elongation could contribute to rodlet bundling, whereby rodlets tend to form oriented domain structures, and our Monte Carlo simulations confirmed this. Surface-catalyzed elongation may be a universal concept to explain the hierarchical assembly mechanism of amyloid fibrils, so it could contribute to the advancement of amyloid research in general.

biophysics↗

bioRxiv2025

Nocturnal intraocular pressure rise is regulated by norepinephrine via RHOB

Intraocular pressure (IOP), a key factor in glaucoma development, is regulated by aqueous humor (AH) dynamics, with inflow from the ciliary body and outflow through the trabecular meshwork (TM). IOP has a circadian rhythm entrained by sympathetic norepinephrine (NE) from the superior cervical ganglion. Herein, we investigated its underlying regulatory mechanisms in the TM. Through comprehensive gene expression analysis of human TM cells and mouse eyes, we identified 18 genes upregulated by NE stimulation, including the small GTPase RAS homologous protein B (RHOB). Promoter assays revealed RHOB upregulation via the cyclic adenosine monophosphate (cAMP) response element on its promotor. NE stimulation for 6-9 h increased RHOB level and cellular adhesion, and suppressed liquid permeability in the TM cells, indicating a time-dependent effect. RHOB deficiency increased TM macrophage phagocytosis and eliminated NE-induced suppression of phagocytosis and permeability, whereas RHOB overexpression had the opposite effect. Instillations of RHO or RHO-kinase inhibitors to mice eye reduced nocturnal and NE-induced IOP elevation. Our findings suggest that NE can elevate IOP via RHOB-mediated inhibition of TM phagocytosis, positioning RHOB as a potential glaucoma treatment target and IOP rhythm regulator.

pathology↗

bioRxiv2023

NMDAR Phosphoproteome Controls Synaptic Growth and Learning

In the mammalian brain, NMDA receptors (NMDARs) activation triggers a calcium-dependent signal transduction cascade resulting in postsynaptic remodeling and behavioral learning. However, the phosphoprotein signal flow through this transduction network is poorly understood. Here, we show that NMDAR-dependent phosphorylation drives the assembly of protein signaling complexes that regulate synaptic morphology and behavior. We performed large-scale phosphoproteomic analyses of protein kinase target proteins in successive layers of the signaling network in mouse striatal/accumbal slices. NMDARs activation resulted in the phosphorylation of 194 proteins, including Rho GTPase regulators. CaMKII-mediated phosphorylation of ARHGEF2 increased its RhoGEF activity, thereby activating the RhoA-Rho-kinase pathway. Subsequent phosphoproteomics of Rho-kinase revealed 221 protein targets, including SHANK3. Experimental validation revealed a pathway from NMDAR-dependent calcium influx through CaMKII, ARHGEF2, Rho-kinase, and SHANK3 to coordinate assembly of an actin-tethered postsynaptic complex of SHANK3/NMDAR/PSD95/DLGAP3 for spine growth and aversive learning. These findings show that NMDARs initiate metabolic phosphorylation for learning.

neuroscience↗

bioRxiv2022

Cleavage of α-1,4-Glycosidic Linkages by the Glycosylphosphatidylinositol-Anchored α-Amylase AgtA Decreases the Molecular Weight of Cell Wall α-1,3-Glucan in Aspergillus oryzae

Aspergillus fungi contain -1,3-glucan with a low proportion of -1,4-glucan as a major cell wall polysaccharide. Glycosylphosphatidylinositol (GPI)-anchored -amylases are conserved in Aspergillus fungi. The GPI-anchored -amylase AmyD in Aspergillus nidulans has been reported to directly suppress the biosynthesis of cell wall -1,3-glucan but not to degrade it in vivo. However, the detailed mechanism of cell wall -1,3-glucan biosynthesis regulation by AmyD remains unclear. Here we focused on AoAgtA, which is encoded by the Aspergillus oryzae agtA gene, an ortholog of the A. nidulans amyD gene. Similar to findings in A. nidulans, agtA overexpression in A. oryzae grown in submerged culture decreased the amount of cell wall -1,3-glucan and led to the formation of smaller hyphal pellets in comparison with the wild-type strain. We analyzed the enzymatic properties of recombinant (r)AoAgtA produced in Pichia pastoris and found that it degraded soluble starch, but not linear bacterial -1,3-glucan. Furthermore, rAoAgtA cleaved 3--maltotetraosylglucose with a structure similar to the predicted boundary structure between the -1,3-glucan main chain and a short spacer composed of -1,4-linked glucose residues in cell wall -1,3-glucan. Interestingly, rAoAgtA randomly cleaved only the -1,4-glycosidic bonds of 3--maltotetraosylglucose, indicating that AoAgtA may cleave the spacer in cell wall -1,3-glucan. Consistent with this hypothesis, heterologous overexpression of agtA in A. nidulans decreased the molecular weight (MW) of cell wall -1,3-glucan. These in vitro and in vivo properties of AoAgtA suggest that GPI-anchored -amylases can degrade the spacer -1,4-glycosidic linkages in cell wall -1,3-glucan before its insolubilization, and this spacer cleavage decreases the MW of cell wall -1,3-glucan in vivo.

microbiology↗

bioRxiv2021

A glycosylphosphatidylinositol-anchored α-amylase encoded by amyD contributes to a decrease in the molecular mass of cell wall α-1,3-glucan in Aspergillus nidulans

-1,3-Glucan is one of the main polysaccharides in the cell wall of Aspergillus nidulans. We previously revealed that it plays a role in hyphal aggregation in liquid culture, and that its molecular mass (MM) in an agsA-overexpressing (agsAOE) strain was larger than that in an agsB-overexpressing (agsBOE) strain. The mechanism that regulates the MM of -1,3-glucan is poorly understood. Although the gene amyD, which encodes glycosyl-phosphatidylinositol (GPI)-anchored -amylase (AmyD), is involved in the biosynthesis of -1,3-glucan in A. nidulans, how it regulates this biosynthesis remains unclear. Here we constructed strains with disrupted amyD ({Delta}amyD) or overexpressed amyD (amyDOE) in the genetic background of the ABPU1 (wild-type), agsAOE, or agsBOE strain, and characterized the chemical structure of -1,3-glucans in the cell wall of each strain, focusing on their MM. The MM of -1,3-glucan from the agsBOE amyDOE strain was smaller than that in the parental agsBOE strain. In addition, the MM of -1,3-glucan from the agsAOE {Delta}amyD strain was greater than that in the agsAOE strain. These results suggest that AmyD is involved in decreasing the MM of -1,3-glucan. We also found that the C-terminal GPI-anchoring region is important for these functions.

microbiology↗

bioRxiv2021

Systematic identification of intron retention associated variants from massive publicly available transcriptome sequencing data

Many disease-associated genomic variants disrupt gene function through abnormal splicing. With the advancement of genomic medicine, identifying disease-associated splicing associated variants has become more important than ever. Most bioinformatics approaches to detect splicing associated variants require both genome and transcriptomic data. However, there are not many datasets where both of them are available. In this study, we developed a methodology to detect genomic variants that cause splicing changes (more specifically, intron retention), using transcriptome sequencing data alone. After demonstrating its high sensitivity and precision, we have applied it to 230,988 transcriptome sequencing data from the publicly available repository and identified 27,937 intron retention associated variants (IRAVs). In addition, by exploring positional relationships with variants registered in existing disease databases, we extracted 3,077 putative disease-associated IRAVs, which range from cancer drivers to variants linked with autosomal recessive disorders. The new in-silico screening framework proposed here provides a foundation for a platform that can automatically acquire medical knowledge making the most of massively accumulated publicly available sequencing data. Collections of IRAVs identified in this study are available through IRAVDB (https://iravdb.io/).

bioinformatics↗

bioRxiv2019

Both galactosaminogalactan and α-1,3-glucan contribute to aggregation of Aspergillusoryzae hyphae in liquid culture

Filamentous fungi generally form aggregated hyphal pellets in liquid culture. We previously reported that -1,3-glucan-deficient mutants of Aspergillus nidulans did not form hyphal pellets and their hyphae were fully dispersed, and we suggested that -1,3-glucan functions in hyphal aggregation. Yet, Aspergillus oryzae -1,3-glucan-deficient (AG{Delta}) mutants still form small pellets; therefore, we hypothesized that another factor responsible for forming hyphal pellets remains in these mutants. Here, we identified an extracellular matrix polysaccharide galactosaminogalactan (GAG) as such a factor. To produce a double mutant of A. oryzae (AG-GAG{Delta}), we disrupted the genes required for GAG biosynthesis in an AG{Delta} mutant. Hyphae of the double mutant were fully dispersed in liquid culture, suggesting that GAG is involved in hyphal aggregation in A. oryzae. Addition of partially purified GAG fraction to the hyphae of the AG-GAG{Delta} strain resulted in formation of mycelial pellets. Acetylation of the amino group in galactosamine of GAG weakened GAG aggregation, suggesting that hydrogen bond formation by this group is important for aggregation. Genome sequences suggest that -1,3-glucan, GAG, or both are present in many filamentous fungi and thus may function in hyphal aggregation in these fungi. We also demonstrated that production of a recombinant polyesterase, CutL1, was higher in the AG-GAG{Delta} strain than in the wild-type and AG{Delta} strains. Thus, controlling hyphal aggregation factors of filamentous fungi may increase productivity in the fermentation industry.\n\nImportanceProduction using filamentous fungi is an important part of the fermentation industry, but hyphal aggregation in these fungi in liquid culture limits productivity compared with that of yeast or bacterial cells. We found that galactosaminogalactan and -1,3-glucan both function in hyphal aggregation in Aspergillus oryzae, and that the hyphae of a double mutant deficient in both polysaccharides become fully dispersed in liquid culture. We also revealed the relative contribution of -1,3-glucan and galactosaminogalactan to hyphal aggregation. Recombinant protein production was higher in the double mutant than in the wild-type strain. Our research provides a potential technical innovation for the fermentation industry that uses filamentous fungi, as regulation of the growth characteristics of A. oryzae in liquid culture may increase productivity.

cell biology↗

bioRxiv2018

Impaired hematopoiesis and leukemia development in mice with a \"knock-in\" allele of U2af1(S34F)

Mutations affecting the spliceosomal protein U2AF1 are commonly found in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (sAML). We have generated mice that carry Cre-dependent \"knock-in\" alleles of U2af1(S34F), the murine version of the most common mutant allele of U2AF1 encountered in human cancers. Cre-mediated recombination in murine hematopoietic lineages caused changes in RNA splicing, as well as multilineage cytopenia, macrocytic anemia, decreased hematopoietic stem and progenitor cells, low-grade dysplasias, and impaired transplantability, but without lifespan shortening or leukemia development. In an attempt to identify U2af1(S34F)-cooperating changes that promote leukemogenesis, we combined U2af1(S34F) with Runx1 deficiency in mice and further treated the mice with a mutagen, N-Ethyl-N-Nitrosourea (ENU). Overall, three of sixteen ENU-treated compound transgenic mice developed AML. However, AML did not arise in mice with other genotypes or without ENU treatment. Sequencing DNA from the three AMLs revealed somatic mutations homologous to those considered to be drivers of human AML, including predicted loss-or gain-of-function mutations in Tet2, Gata2, Idh1, and Ikzfl. However, the engineered U2af1(S34F) missense mutation reverted to wild type (WT) in two of the three AML cases, implying that U2af1(S34F) is dispensable, or even selected against, once leukemia is established.\n\nSIGNIFICANCE STATEMENTSomatic mutations in four splicing factor genes (U2AF1, SRSF2, SF3B1, and ZRSR2) are found in MDS and MDS-related AML, blood cancers with few effective treatment options. However, the pathophysiological effects of these mutations remain poorly characterized, in part due to the paucity of disease-relevant models. Here, we report the establishment of mouse models to study the most common U2AF1 mutation, U2af1(S34F). Production of the mutant protein specifically in the murine hematopoietic compartment disrupts hematopoiesis in ways resembling human MDS. We further identified deletion of the Runx1 gene and other known oncogenic mutations as changes that might collaborate with U2af1(S34F) to give rise to frank AML in mice.

cancer biology↗

bioRxiv2017

ProteomeGenerator: A framework for comprehensive proteomics based on de novo transcriptome assembly and high-accuracy peptide mass spectral matching

Modern mass spectrometry now permits genome-scale and quantitative measurements of biological proteomes. However, analyses of specific specimens are currently hindered by the incomplete representation of biological variability of protein sequences in canonical reference proteomes, and the technical demands for their construction. Here, we report ProteomeGenerator, a framework for de novo and reference-assisted proteogenomic database construction and analysis based on sample-specific transcriptome sequencing and high-resolution and high-accuracy mass spectrometry proteomics. This enables assembly of proteomes encoded by actively transcribed genes, including sample-specific protein isoforms resulting from non-canonical mRNA transcription, splicing, or editing. To improve the accuracy of protein isoform identification in non-canonical proteomes, ProteomeGenerator relies on statistical target-decoy database matching augmented with spectral-match calibrated sample-specific controls. We applied this method for the proteogenomic discovery of splicing factor SRSF2-mutant leukemia cells, demonstrating high-confidence identification of non-canonical protein isoforms arising from alternative transcriptional start sites, intron retention, and cryptic exon splicing, as well as improved accuracy of genome-scale proteome discovery. Additionally, we report proteogenomic performance metrics for the current state-of-the-art implementations of SEQUEST HT, Proteome Discoverer, MaxQuant, Byonic, and PEAKS mass spectral analysis algorithms. Finally, ProteomeGenerator is implemented as a Snakemake workflow, enabling open, scalable, and facile discovery of sample-specific, non-canonical and neomorphic biological proteomes (https://github.com/jtpoirier/proteomegenerator).

bioinformatics↗