Biology subjects

Fukuta, T.

Publications and source records attributed to Fukuta, T..

3 recordsLinked to original sources

bioRxiv2025

Emergence of a Cell-Guided Multivalent Ligand of Enzymes on Cancer Cells Triggered by Click Reaction between Hetero Nano-Assemblies

Stimuli-responsive nanomaterials with multivalent ligands have attracted significant attention in the context of cancer chemotherapy and imaging. However, challenges remain such as non-selective stimuli response and/or existence of triggers in healthy regions in addition to the intrinsic heterogeneity of cancer that causes insufficient target recognition. In-spired by the expression of precise and diverse functions of biological machineries triggered by specific protein-protein complexation and conformational change, we report an artificial system where a bioorthogonal click reaction between hetero-nano-assemblies triggers their complexation and conformational changes, resulting in emergence of multivalent ligands for cancer-associated enzymes: carbonic anhydrase IX (CAIX). We also demonstrated that the multivalent ligands selectively inhibited the proliferation of cancer cells overexpressing CAIX under hypoxic conditions. Additionally, the click reaction between nano-assemblies in the presence of target cells provided a higher efficacy of the emerged multivalent ligands than that pre-formed in the absence of cells. Our study provides a basis for the development of multivalent ligands displaying adaptive binding interfaces for target cancer cells with high selectivity and affinity to thus potentially overcome tumor heterogeneity.

biochemistry↗

bioRxiv2024

Lipid- and protein-directed photosensitizer proximity labeling captures the cholesterol interactome

The physical properties of cellular membranes, including fluidity and function, are influenced by protein and lipid interactions. In situ labeling chemistries, most notably proximity-labeling interactomics are well suited to characterize these dynamic and often fleeting interactions. Established methods require distinct chemistries for proteins and lipids, which limits the scope of such studies. Here we establish a singlet-oxygen-based photocatalytic proximity labeling platform (POCA) that reports intracellular interactomes for both proteins and lipids with tight spatiotemporal resolution using cell-penetrant photosensitizer reagents. Using both physiologically relevant lipoprotein-complexed probe delivery and genetic manipulation of cellular cholesterol handling machinery, cholesterol-directed POCA captured established and unprecedented cholesterol binding proteins, including protein complexes sensitive to intracellular cholesterol levels and proteins uniquely captured by lipoprotein uptake. Protein-directed POCA accurately mapped known intracellular membrane complexes, defined sterol-dependent changes to the non-vesicular cholesterol transport protein interactome, and captured state-dependent changes in the interactome of the cholesterol transport protein Aster-B. More broadly, we find that POCA is a versatile interactomics platform that is straightforward to implement, using the readily available HaloTag system, and fulfills unmet needs in intracellular singlet oxygen-based proximity labeling proteomics. Thus, we expect widespread utility for POCA across a range of interactome applications, spanning imaging to proteomics.

biochemistry↗

bioRxiv2021

Distinct repertoire expansion patterns during affinity maturation of antigen responding camelid-derived single-domain VHH antibody

Antibody maturation is the central function of the adaptive immune response. This process is driven by the repetitive selection of mutations that increase the affinity toward antigens. We hypothesized that precise observation of this process by high-throughput sequencing along time course of immunization will enable us to predict the antibodies reacting to the immunized antigen without any additional in vitro screening. An alpaca was immunized by IgG fragments using multiple antigen injections and antibody repertoire development was traced via high-throughput sequencing periodically for months. The sequences were processed into clusters and the antibodies in the 16 most abundant clusters were generated to determine whether the clusters included antigen-binding antibodies. The sequences of most antigen-responsive clusters resembled those of germline cells in the early stages. These sequences were observed to accumulate significant mutations and also showed continuous sequence turnover throughout the experimental period. The foregoing characteristics gave us 75% successful prediction of clusters composed of antigen-responding VHHs against IgG fragment. In addition, the prediction method was applied to the data from other alpaca immunized by epidermal growth factor receptor and 77% successful prediction confirmed the generality of the prediction method. Superior to previous studies, we identified the immune responsive but very rare clusters or sequences from the immunized alpaca without any empirical screening data. Significance StatementWe have developed a method for selecting VHH antibody sequences that react to antigens without in vitro screening by performing a large-scale sequence analysis of VHH along the immunization time course, clustering the sequences, and tracking the evolution of the sequences in the clusters. This method enables the identification of antibodies with low frequencies of occurrence, which has been difficult with existing in silico antibody identification methods.

immunology↗