Biology subjects

Minami, A.

Publications and source records attributed to Minami, A..

8 recordsLinked to original sources

bioRxiv2025

Generative AI-based design of hybrid transcriptional activator proteins with new DNA-binding specificity

Transcriptional control arises from the specific recognition of promoter DNA by transcription factors (TFs), forming the basis of cellular information processing and gene regulation. In synthetic biology, TF-promoter interactions are assembled into genetic circuits to program cellular behaviors. To ensure expected circuit performance, most synthetic genetic circuits rely on combinations of well-characterized and orthogonal transcriptional regulations. This reliance minimizes crosstalk but constrains circuit complexity and information integration. Creating hybrid TFs that combine or interpolate promoter specificities could therefore expand the design space of synthetic regulatory systems. However, it remains unclear whether hybrid functions can be created by mixing amino acid sequences, and how such functional integration could be achieved in a principled manner. Here we show that a variational autoencoder (VAE) trained on LuxR-family DNA-binding domains can generate transcription factors with hybrid and partially novel promoter recognition properties. By sampling intermediate regions of the VAE-learned latent space, we designed hybrid TFs that activate both the lux and las promoters. High-throughput sort-seq assays together with individual in vivo assays revealed that a subset of functional variants exhibited dual-responsive behavior while maintaining sequence-selective DNA recognition. Together, these results provide a data-driven strategy for exploring functional intermediate sequences between closely related proteins.

synthetic biology↗

bioRxiv2025

Predicting protein complexes in biosynthetic gene clusters

Biosynthetic gene clusters (BGCs) are contiguous genomic regions that encode diverse proteins responsible for natural product biosynthesis. These proteins collectively produce various secondary metabolites with complex chemical structure, including antibiotics and mycotoxins, yet the complete biosynthetic pathways have been experimentally resolved for only a limited number of compounds. Protein-protein interactions within BGCs have recently been recognized as key determinants of intermediate transfer, enzymatic regulation, and structural stability. However, many BGCs still contain proteins of unknown function that cannot be predicted by conventional sequence-based bioinformatics tools, hindering a comprehensive understanding of their biosynthetic pathways. To address this challenge, we built a high-throughput complex prediction pipeline by replacing AlphaFold3s multiple sequence alignment generation with a faster MMSeqs2. We systematically screened 487,828 protein pairs derived from 2,437 BGCs registered in the Minimum Information about a Biosynthetic Gene cluster (MIBiG) database and predicted 15,438 heteromeric interactions with an ipTM [≥] 0.6. Among them, 1,390 protein pairs exhibited structural homology with an RMSD [≤] 2.0 [A]. These predictions highlight interesting molecular mechanisms involving proteins previously annotated as "uncharacterized" or "potentially dysfunctional". Our analysis further showed that the ipSAE metric can distinguish correct heterocomplex pairs when multiple functionally homologous proteins are present within a BGC. Overall, our computational analysis revealed molecular interaction networks among proteins encoded by each BGC and identified enzyme complexes that are likely functional only when assembled. These predicted complexes may represent previously unrecognized links in their biosynthetic pathways. The complete results are available in a reusable format at https://doi.org/10.5281/zenodo.17451667 to support future experimental validation.

bioinformatics↗

bioRxiv2025

Differential O-Glc elongation on the specific EGF repeat within a ligand binding domain regulates NOTCH1 signaling

Three types of O-linked glycosylation - O-glucose, O-fucose, and O-N-acetylglucosamine - are crucial for the function of Notch receptors, which regulates critical cell fate determination processes in a wide variety of contexts. O-Glucose glycans are added to serine residues located between the first and second conserved cysteines within the epidermal growth factor (EGF) -like repeats in the Notch extracellular domain. Previously, O-glucose glycans were shown to be extended to a trisaccharide structure with two xyloses via an 1-3 linkage. Our recent studies, however, indicated that the O-glucose glycan on NOTCH1 EGF10 can be extended by hexose and Neu5Ac. Here, we demonstrated that this hexose- and Neu5Ac-extended glycan has a 3-sialyllactose-like structure synthesized by specific members of two isoenzyme families, B4GALT1 and ST3GAL4. Using mass spectrometry, we identified this modification exclusively on NOTCH1 EGF10 and NOTCH3 EGF9 (equivalent to NOTCH1 EGF10), with no detection in any other EGF domains in NOTCH1-3. Sequence comparison and mutagenesis experiments identified one amino acid at position -2 of the fourth cysteine in the EGF domain as crucial for the galactose elongation of O-glucose glycans. We further demonstrated that this site-specific elongation of O-glucose on NOTCH1 EGF10 significantly impact ligand binding and signal transduction of NOTCH1. Our findings contribute to the understanding of the intricate regulatory mechanisms of Notch receptor function mediated by distinct positions and structures of O-glycans. Significance StatementNotch, a signaling receptor critical for development, differentiation, and homeostasis in multicellular organisms, undergoes multiple post-translational glycan modifications to regulate its function. While O-glucose glycan elongation by xylose was previously known, we identified an O-glucose glycan extended by galactose and Neu5Ac on specific epidermal growth factor (EGF) -like repeats of certain Notch paralogs, NOTCH1 and NOTCH3. As far as we know, this structure has not been reported on any other proteins. We identify the enzymes involved in the biosynthesis of this glycan and demonstrate that modifications at a specific site on Notch direct receptor functions, including ligand binding and Notch signaling.

biochemistry↗

bioRxiv2024

Tuning of ribosome levels mediated by RNase I and hibernating ribosomes

Protein biosynthesis is an energy-hungry intracellular process that requires the stringent regulation of ribosome abundance under environmental conditions. In response to stress, some active ribosomes are degraded while others, in bacteria, enter a hibernation state to protect against degradation. RNase I, a conserved T2 family ribonuclease in Escherichia coli, degrades ribosomal RNA to suppress biofilm formation, whereas it interacts with ribosomes. However, how and why RNase I binds to ribosomes remains elusive. Here, we show that hibernating ribosomes bind to RNase I and inhibit its activity, thereby promoting biofilm formation. We determined the cryo-electron microscopy structure of the hibernating ribosome complexed with RNase I. RNase I interacts with helix 41 of the 16S rRNA and the ribosomal protein uS14 in the head domain of the 30S subunit of the hibernating ribosome and positions its active centre away from helix 41, resulting in its catalytic inactivation. Hibernating ribosomes are protected from RNase I-mediated cleavage, and our in vivo and in vitro analyses revealed that RNase I targets dissociated large and small ribosomal subunits for rRNA degradation. These findings reveal a previously uncharacterized regulatory strategy that ribosomes modulate RNase I activity, ensuring both the preservation and timely degradation of ribosomes during environmental stress adaptation.

molecular biology↗

bioRxiv2024

A fungal transcription factor BOT6 facilitates the transition of a beneficial root fungus into an adapted anthracnose pathogen

The infection strategies employed by plant endophytes are attributed to their ability to overcome durable nonhost resistance and adapt to the host environment. However, the regulatory genetic background underlying how they adapt to the host and determine their lifestyles remains enigmatic. Here, we show that the CtBOT6, a cluster-residing transcription factor in the root-associated fungus Colletotrichum tofieldiae (Ct), plays a pivotal role in regulating virulence-related gene expression and in producing metabolites both not only within and but unexpectedly outside of the cluster. Genetic manipulation of CtBOT6 toward activation alone is sufficient to transition a root beneficial Ct along the mutualist-pathogen continuum even toward a leaf pathogen capable of overcoming nonhost resistance, partly dependent on the host abscisic acid and ethylene pathways. Our findings indicate that the status of CtBOT6 serves as a critical determinant for the endophytic fungus to adapt to the plant different environments and manifest diverse infection strategies.

plant biology↗

bioRxiv2023

Relationship Between Domain and Function of the Yeast RNase T2, Rny1p, Which Mediates rRNA Degradation upon Starvation

RNase T2 is ubiquitous across diverse organisms, playing essential roles despite its simple enzymatic activity. In Saccharomyces cerevisiae, RNase T2, known as Rny1p, localizes in vacuoles and mediates rRNA degradation during autophagy of ribosomes. In this study, we elucidated novel aspects of ribosome degradation mechanisms and the function of Rny1p. First, we discovered that most ribosomes are degraded by selective autophagy, where Rsa1p is the specific receptor of ribosomes to be degraded. Complex structure prediction suggested that Rsa1p also interacts with Atg8p. Furthermore, we observed that the accumulation of rRNA in vacuoles, due to the lack of Rny1p, leads to a decrease in bulk autophagic activity. This decrease in autophagic activity may explain the inability of Rny1p-deficient strains to adapt to starvation conditions. Second, our structural prediction and biochemical analyses indicate that a C-terminal extension, characteristic in fungal RNase T2 including Rny1p, is not necessary for rRNA degradation but for anchoring to the cell wall. Together with molecular phylogenetic analysis, a species-specific role of RNase T2 conferred by the C-terminal extension is suggested.

molecular biology↗

bioRxiv2021

Convergent evolution of a novel blood-red nectar pigment in vertebrate-pollinated flowers

Nesocodon mauritianus (Campanulaceae) produces a blood-red nectar that has been proposed to serve as a visual attractant for pollinator visitation. Here we show that the nectars red color is derived from a novel alkaloid termed nesocodin. The first nectar produced is acidic and pale yellow in color, but slowly becomes alkaline before taking on its characteristic red color. Three enzymes secreted into the nectar are either necessary or sufficient for pigment production, including (1) a carbonic anhydrase that creates an alkaline environment, (2) an aryl alcohol oxidase that generates sinapaldehyde, a pigment precursor, and (3) a ferritin-like catalase that protects nesocodin from degradation by hydrogen peroxide. Our findings demonstrate how these three enzymatic activities allow for the condensation of sinapaldehyde and proline to form a novel pigment with a stable imine bond, which in turn is attractive to Phelsuma geckos, the presumed pollinators of Nesocodon. We also identify nesocodin in the red nectar of the distantly related Jaltomata herrerae and provide evidence for convergent evolution of this trait. While the overall enzymatic activities required for red pigment formation in both Nesocodon and J. herrerae nectars are identical, the associated genes encoding the enzymes are not orthologous and, in the case of the aryl alcohol oxidase, even belong to different protein families. This work cumulatively identifies a novel, convergently evolved trait in two vertebrate-pollinated species, suggesting the red pigment is selectively favored and that only a limited number of compounds are likely to underlie this adaptation. SignificanceNearly 90% of flowering plants produce nectar to attract pollinators. Beyond sugars, many types of nectar solutes play important ecological roles; however, the molecular basis for the diversity of nectar composition across species is less explored. One rare trait among flowering plants is the production of colored nectar, which may function to attract and guide prospective pollinators. Our findings indicate convergent evolution of a red-colored nectar across two distantly related plant species. Behavioral data show that the red pigment attracts diurnal geckos, a presumed pollinator of one of these plants. These findings join a growing list of examples of distinct biochemical and molecular mechanisms underlying evolutionary convergence, and provide a fascinating system for testing how interactions across species drive the evolution of novel pigments in an understudied context.

evolutionary biology↗

bioRxiv2021

A cell wall invertase controls nectar volume and sugar composition

Flowering plants produce nectar to attract pollinators. The main nectar sugars are sucrose, glucose, and fructose, which can vary widely in ratio and concentration across species. Brassica spp. produce a hexose-dominant nectar (high in the monosaccharides glucose and fructose) with very low levels of the disaccharide sucrose. Cell wall invertases (CWINVs) catalyze the irreversible hydrolysis of sucrose into glucose and fructose in the apoplast. We found that BrCWINV4A is highly expressed in the nectaries of Brassica rapa. Moreover, a brcwinv4a null mutant has (1) greatly reduced cell wall invertase activity in the nectaries, and (2) produces a sucrose-rich nectar with little hexose content, but (3) with significantly less volume. These results were recapitulated via exogenous application of an invertase inhibitor to wild-type flowers. Honeybees prefer nectars with some sucrose, but wild-type B. rapa flowers were much more heavily visited than those of brcwinv4a, suggesting that the potentially attractive sucrose-rich nectar of brcwinv4a could not compensate for its low volume. These results cumulatively indicate that BrCWINV4A is not only essential for producing a hexose-rich nectar, but also support a model of nectar secretion in which its hydrolase activity is required for maintaining a high intracellular-to-extracellular sucrose ratio that facilitates the continuous export of sucrose into the apoplast via SWEET9. Extracellular hydrolysis of each sucrose into two hexoses by BrCWINV4A also likely creates the osmotic potential required for nectar droplet formation. In summary, modulation of CWINV activity can at least partially account for naturally occurring differences in nectar volume and sugar composition.

plant biology↗