Biology subjects

Kamiya, M.

Publications and source records attributed to Kamiya, M..

8 recordsLinked to original sources

bioRxiv2026

Enzyme activity as an actionable axis for small-molecule precision oncology

Current precision oncology--molecular targeted therapies and immunotherapies--relies on genomic or expressed biomarkers, yet most cancer patients remain ineligible for these treatments. Here, we establish enzyme activity as an actionable and orthogonal axis for precision cancer medicine. Strategic activity-based screening of mouse organs and human clinical specimens with a panel of enzyme-reactive fluorescence probes identified {beta}-galactosidase 1 (GLB1) and {beta}-hexosaminidases (HEX) as broadly elevated tumor-selective biomarkers. Leveraging these activities, we developed 7-ethyl-10-hydroxycamptothecin (SN38)-based GLB1-and HEX-reactive prodrugs. These prodrugs exhibited dramatically reduced systemic toxicities and improved therapeutic windows, compared to a clinically used SN38-based prodrug, irinotecan (CPT-11). Both prodrugs demonstrated activity-dependent therapeutic efficacy, affording a dramatic reduction of tumor volumes across multiple in vivo models, including a subcutaneous patient-derived xenograft (PDX) of lung squamous cell carcinoma that lacked genetic alterations targeted by current precision medicine. Furthermore, this strategy is broadly applicable across various cytotoxic payloads, establishing a generalizable platform for small-molecule precision medicines. Our results define an enzyme-targeting paradigm for precision oncology, in which fluorescence probes serve as companion diagnostic tools to guide development and selection of appropriately targeted prodrugs, which are expected to provide safer and more efficacious treatment options for cancer patients with elevated enzyme activities.

cancer biology↗

bioRxiv2025

TGF-β Coordinates Alanine Synthesis and Import for Myofibroblast Differentiation in Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease marked by aberrant fibroblast-to-myofibroblast differentiation, a process that requires metabolic reprogramming. We identify alanine as a critical metabolite that confers metabolic flexibility to support differentiation. TGF-{beta} increases alanine by activating both its synthesis and import in normal and IPF lung fibroblasts. Alanine is synthesized primarily by GPT2, which is regulated by a glutamine-glutamate--ketoglutarate axis. Inhibiting GPT2 depletes alanine and suppresses TGF-{beta}-induced expression of -SMA and COL1A1, an effect reversed by alanine supplementation. We also identify SLC38A2 as a key transporter of both alanine and glutamine that is upregulated by TGF-{beta} and alanine deprivation. Together, SLC38A2 and GPT2 activities converge to maintain intracellular alanine levels to support myofibroblast differentiation. Mechanistically, alanine deficiency suppresses glycolysis and depletes tricarboxylic acid cycle intermediates, while supplementation provides carbon and nitrogen for intracellular glutamate and proline biosynthesis, particularly in the absence of glutamine. Combined inhibition of GPT2 and SLC38A2 suppresses fibrogenic responses in fibroblasts and in human precision-cut lung slices, highlighting a potential therapeutic strategy for fibrotic lung disease.

cell biology↗

bioRxiv2025

Low-Background Cancer Imaging With a Bioorthogonal Fluorescence Probe and Engineered Reporter Enzyme Bearing a Targeting Moiety

Combinatorial use of an antibody-reporter enzyme conjugate and a fluorescence probe activated by the enzyme is a powerful strategy for fluorescence-guided cancer surgery. However, conventional probes for typical reporter enzymes are insufficiently bioorthogonal, resulting in high background signals in non-target tissues. We screened a library of HMRef (rhodol derivative)-based fluorescence probes bearing various sugar moieties, and discovered that HMRef-{beta}-D-fucose is bioorthogonal in mammalian systems, but is activated by a metagenomic glycosidase, Td2F2. Directed evolution generated a mutant with a kcat/Km value for HMRef-{beta}-D-fucose of 3.3 x 105/M/sec, 7.3 times higher than that of wild-type Td2F2 and comparable to that of {beta}-galactosidase (LacZ) with a corresponding probe. Theoretical calculation suggested that E296G mutation in Td2F2 causes structural changes that facilitate the probes access to the enzymes active site. In a proof-of-concept study, cancer cells were visualized with a minimal background in the mesentery of a mouse model of peritoneally disseminated human-ovarian-cancer-derived SKOV-3 cells, which endogenously express HER2, by using HMRef-{beta}-D-fucose together with engineered Td2F2 conjugated/fused to a HER2-binding antibody/nanobody.

bioengineering↗

bioRxiv2025

A CD57+ cytotoxic CD8 T cell subset associated with fibrotic lung disease in systemic sclerosis

Interstitial lung disease (ILD) is a major cause of morbidity and mortality in systemic sclerosis (SSc); however, the immunopathologic mechanisms driving lung disease in SSc are unclear. T cells have been implicated as a likely driver of lung injury in SSc. Here, we have evaluated T cells in blood and lungs of patients with SSc-ILD and identified a specific population of cytotoxic CD8 T cells that is expanded in SSc-ILD patients. Cytotoxic effector memory CD8 T cells marked by CD57 expression are preferentially expanded in SSc-ILD patients compared to SSc patients without ILD and controls and show prominent clonal expansion. These CD57+ T effector memory (TEM) cells differ from T effector memory cells re-expressing CD45RA (TEMRA) transcriptomically and functionally, with cytotoxic function that is enhanced by CD155 engagement of the costimulatory receptor CD226. Analyses of cells from ILD lungs indicate endothelial cells as a likely source of CD155 to activate CD57+ cytotoxic T cells. Together, the results implicate a CD57+ cytotoxic CD8 T cell population as a potential mediator of lung injury in SSc-ILD.

immunology↗

bioRxiv2023

Barcoding of small extracellular vesicles with CRISPR-gRNA enables comprehensive, subpopulation-specific analysis of their biogenesis/release regulators

Small extracellular vesicles (sEVs) are important intercellular information transmitters in various biological contexts, but their release processes remain poorly understood. Herein, we describe a high-throughput assay platform, CRISPR-assisted individually barcoded sEV-based release regulator (CIBER) screening, for identifying key players in sEV release. CIBER screening employs sEVs barcoded with CRISPR-gRNA through the interaction of gRNA and dead Cas9 fused with an sEV marker. Barcode quantification enables the estimation of the sEV amount released from each cell in a massively parallel manner. Barcoding sEVs with different sEV markers in a CRISPR pooled-screening format allows genome-wide exploration of sEV release regulators in a subpopulation-specific manner, successfully identifying previously unknown sEV release regulators and uncovering the exosomal/ectosomal nature of CD63+/CD9+ sEVs, respectively, as well as the synchronization of CD9+ sEV release with the cell cycle. CIBER should be a valuable tool for detailed studies on the biogenesis, release, and heterogeneity of sEVs.

cell biology↗

bioRxiv2022

Super-resolution vibrational imaging based on photoswitchable Raman probe

Super-resolution vibrational microscopy is a promising tool to increase the degree of multiplexing of nanometer-scale biological imaging, because the spectral linewidth of molecular vibration is about 50 times narrower than that of fluorescence. However, current techniques of super-resolution vibrational microscopy still suffer from various limitations including the need for cell fixation, high power loading or complicated frequency-modulated detection schemes. Herein we utilize photoswitchable stimulated Raman scattering (SRS) to develop a method that we call reversible saturable optical Raman transitions (RESORT) microscopy, which overcomes these limitations. We first describe a new kind of photoswitchable Raman probe designated DAE620 and then we employ a standard SRS detection scheme to validate its signal activation and depletion characteristics when exposed to low-power (microwatt level) continuous-wave laser light. By harnessing the SRS signal depletion of DAE620 through a donut-shaped beam, we demonstrate super-resolution vibrational imaging of mammalian cells with excellent chemical specificity and spatial resolution beyond the optical diffraction limit. Our results indicate RESORT microscopy to be an effective tool with high potential for multiplexed super-resolution imaging of live cells.

bioengineering↗

bioRxiv2021

Near-infrared imaging in fission yeast by genetically encoded biosynthesis of phycocyanobilin

Near-infrared fluorescent protein (iRFP) is a bright and stable fluorescent protein with excitation and emission maxima at 690 nm and 713 nm, respectively. Unlike the other conventional fluorescent proteins such as GFP, iRFP requires biliverdin (BV) as a chromophore because iRFP originates from bacteriophytochrome. Here, we report that phycocyanobilin (PCB) functions as a brighter chromophore for iRFP than BV, and biosynthesis of PCB allows live-cell imaging with iRFP in the fission yeast Schizosaccharomyces pombe. We initially found that fission yeast cells did not produce BV, and therefore did not show any iRFP fluorescence. The brightness of iRFP attached to PCB was higher than that of iRFP attached to BV in vitro and in fission yeast. We introduced SynPCB, a previously reported PCB biosynthesis system, into fission yeast, resulting in the brightest iRFP fluorescence. To make iRFP readily available in fission yeast, we developed an endogenous gene tagging system with iRFP and all-in-one integration plasmids, which contain genes required for the SynPCB system and the iRFP-fused marker proteins. These tools not only enable the easy use of iRFP in fission yeast and the multiplexed live-cell imaging in fission yeast with a broader color palette, but also open the door to new opportunities for near-infrared fluorescence imaging in a wider range of living organisms.

cell biology↗

bioRxiv2020

Self-labeling of proteins with chemical fluorescent dyes in BY-2 cells and Arabidopsis seedlings

AO_SCPLOWBSTRACTC_SCPLOWSynthetic chemical fluorescent dyes are promising tools for many applications in biology. SNAP tagging provides a unique opportunity for labeling of specific proteins in vivo with synthetic dyes for studying for example endocytosis, or super-resolution microscopy. However, despite the potential, chemical dye tagging has not been used effectively in plants. A major drawback was the limited knowledge regarding cell wall and membrane permeability of synthetic dyes. Twenty-six out of 31 synthetic dyes were taken up into BY-2 cells, eight were not taken up and can thus serve for measuring endocytosis. Three of the dyes that were able to enter the cells, SNAP-tag ligands of diethylaminocoumarin, tetramethylrhodamine (TMR) and silicon-rhodamine (SiR) 647 were used to SNAP tag -tubulin. Successful tagging was verified by live cell imaging and visualization of microtubules arrays in interphase and during mitosis. Fluorescence activation-coupled protein labeling (FAPL) with DRBG-488 was used to observe PIN2 endocytosis and delivery to the vacuole as well as preferential delivery of newly synthesized PIN2 to the newly forming cell plate during mitosis. Together the data demonstrate that specific self-labeling of proteins can be used effectively in plants to study a wide variety to cell biological processes.

cell biology↗