Biology subjects

Komatsu, T.

Publications and source records attributed to Komatsu, T..

16 recordsLinked to original sources

bioRxiv2026

Biosensor-enabled single-molecule enzyme activity profiling for function-based molecular counting

Single-molecule enzyme activity analysis enables proteoform-resolved quantification of catalytically active enzyme molecules but remains limited by the need for enzyme-specific fluorogenic substrates, particularly for enzymes involved in central metabolism. Here we establish a biosensor-based framework that integrates genetically encoded fluorescent metabolite biosensors with single-molecule enzyme activity profiling (SEAP), enabling native enzymatic reactions to be monitored without enzyme-specific fluorogenic substrates. By employing different biosensors, the framework was applied to distinct metabolic enzymes, including lactate dehydrogenase and pyruvate kinase. Biosensor-based SEAP enables absolute quantification of active enzyme molecules, transforming conventional activity measurements (U/mL) into molecular abundance (molecules/mL). Applying this approach to single-cell functional proteoform analysis revealed functional heterogeneity in lactate dehydrogenase activity during T-cell activation. In addition, analysis of circulating enzymes showed that much of the plasma lactate dehydrogenase pool could be quantitatively accounted for by physiological erythrocyte turnover. These results establish genetically encoded fluorescent protein-based metabolite biosensors as modular optical transducers for single-molecule functional enzyme analysis across cellular and circulating samples.

biochemistry↗

bioRxiv2026

Topical Activatable Fluorescence Probes for Rapid Intraoperative Detection of Peritoneal Dissemination in High-Grade Serous Ovarian Carcinoma

BackgroundEpithelial ovarian cancer (EOC) is one of the most lethal gynecologic malignancies, largely because most patients are diagnosed at an advanced stage with peritoneal dissemination. High-grade serous carcinoma (HGSC), the most common and aggressive EOC subtype, requires complete cytoreductive surgery to improve the prognosis; however, minute disseminated lesions are often missed by conventional intraoperative inspection. Here, we aimed to develop fluorescence probes for rapid and sensitive intraoperative detection of HGSC peritoneal dissemination. MethodsWe screened a hydroxymethyl rhodamine green (HMRG)-based fluorescence probe library consisting of 381 protease- and aminopeptidase-reactive fluorescence probes using tumor and non-tumor specimens from patients with HGSC. The target enzyme of the hit probes was identified by means of enzyme assays, immunohistochemistry, and LC/MS analysis. Diagnostic utility was evaluated ex vivo using clinical specimens and in vivo using a peritoneal dissemination mouse model. ResultsThree probes--EK-HMRG, NA-HMRG and DA-HMRG--were selected as promising candidates for the detection of peritoneal dissemination in HGSC. Puromycin-sensitive aminopeptidase (PSA) was identified as a novel target enzyme of these probes. EK-HMRG, NA-HMRG and DA-HMRG rapidly detected peritoneal dissemination just a few millimeters in size with high sensitivity and specificity in clinical HGSC specimens and in a peritoneal dissemination mouse model after topical application. ConclusionsThe PSA-targeting topical fluorescence probes EK-HMRG, NA-HMRG and DA-HMRG are promising tools for real-time, highly sensitive intraoperative visualization of peritoneal dissemination in HGSC, and are promising candidates to improve complete resection rates.

cancer biology↗

bioRxiv2026

Early detection of pancreatic ductal adenocarcinoma by single-molecule profiling of pancreatic enzyme activities

Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer-related mortality, largely due to diagnosis at advanced stages. Early detection through minimally invasive liquid biopsy holds promise for improving patient outcomes. Here, we report a blood-based liquid biopsy platform based on single-molecule enzyme activity profiling (SEAP), which detects proteoform-level alterations in circulating pancreatic enzymes at single-molecule resolution. Using a tissue-centric biomarker discovery strategy, we identified activity signatures of pancreas-specific digestive enzymes associated with PDAC. A combinatorial classifier detected PDAC (stage I-IV) with 95.5% specificity and 75.0% sensitivity (74.2% for stage I-II) across 690 blood samples collected from multiple hospitals and biobanks. Performance was further validated in an independent cohort enriched for early-stage disease, where 54.2% (13/24) of stage IA, 64.3% (9/14) of stage IB, and 21.4% (3/14) of stage 0 lesions were classified as positive. These findings support the clinical potential of SEAP for early detection of PDAC.

biochemistry↗

bioRxiv2026

Single-Molecule Activity Profiling of Glycosidase Proteoforms Using Water-Soluble Fluorogenic Probes

In this study, we report a series of highly water-soluble fluorogenic probes for glycosidases useful for microdevice-based single-molecule enzyme activity profiling (SEAP). The assay was able to detect trace glycosidase activities in blood with high sensitivity and with proteoform resolution. This platform revealed the potential of -mannosidase as sensitive blood-based liquid-biopsy biomarkers for liver injury, highlighting the potential of this approach for activity-based diagnostics.

biochemistry↗

bioRxiv2026

Distribution and p300/CBP dependency of cis-regulatory elements are encoded by unique combinations of histone lactylation and acetylation

Histone lactylation H3K18la marks tissue-specific enhancers in combination with H3K27ac. It remains unclear whether and how these modifications differ. In this study, we dissected the specific roles of H3K18la- and H3K27ac-marked regions. We found that H3K18la predominantly localized at non-promoter regions with lineage-specific transcription factors and was strongly associated with enhancer activity and p300/CBP dependency. In contrast, H3K27ac was largely independent of these features; however, H3K27ac-occupied regions were selectively bound by the super-enhancer-enriched transcriptional coactivators BRD4 and MED1 and were responsible for gene expression involving liquid-liquid phase separation. The genomic regions covered by both H3K18la and H3K27ac best overlapped with super-enhancers. Thus, our study suggests that p300/CBP-dependent enhancer potential and BRD4/MED1-mediated enhancer cooperativity are separately encoded by H3K18la and H3K27ac, respectively, and exert synergistic enhancer activation when combined. Although the characteristics of H3K18la were similar to those of N-terminal acetylation of H2B (H2BNTac), a recently described enhancer signature, we found distinct genomic distributions of these marks. Collectively, our findings will advance the current understanding of how histone modifications establish enhancer landscapes.

molecular biology↗

bioRxiv2026

Solid-Phase Synthesis of ProTide Fluorogenic Probes Enables Systematic Profiling of Carboxypeptidase Activity

Carboxypeptidases play diverse roles in physiological and pathological processes, yet comprehensive analysis of their activities in complex biological samples remains challenging. Here we report a solid-phase synthesis strategy for fluorogenic ProTide-based probes that enables systematic profiling of carboxypeptidase activities based on defined C-terminal amino acid motifs. By modular synthesis of dipeptide-fluorophore conjugates, we generated a focused probe set that revealed distinct substrate preferences among carboxypeptidases, including carboxypeptidase A and B family enzymes. Integration of these probes with a single-molecule enzyme activity assay allowed ultrasensitive detection of circulating carboxypeptidase activities in human blood samples. Application of this platform to clinical specimens demonstrated that specific carboxypeptidase activities are elevated in patients with pancreatic cancer compared with healthy controls, whereas closely related enzymes showed limited diagnostic value. These results establish a scalable chemical strategy for activity-based profiling of exopeptidases and highlight circulating carboxypeptidase activity as a functional enzymatic signature associated with pancreatic cancer.

biochemistry↗

bioRxiv2026

Synthesis based on covalent capture and release (SCCR): a programmable strategy for automated preparation of protease-activatable molecules

Enzyme-activatable chemical tools, including fluorogenic probes and prodrugs, are essential in chemical biology and targeted therapeutics but remain challenging to access in structurally diverse forms because their synthesis is often bespoke and difficult to standardize. Here, we introduce synthesis based on covalent capture and release (SCCR) as a programmable chemical strategy that enables the modular assembly of protease-activatable molecules through specifically designed protecting-group logic. The SCCR framework establishes a standardized capture-elongation- release workflow that decouples molecular diversification from individual synthetic optimization, thereby enabling automated preparation of complex libraries. Using this chemistry, we generated a diverse set of fluorogenic probes and applied them to single-molecule enzyme activity analyses to identify candidate activity-based biomarkers of liver diseases. The generality of the SCCR strategy was further demonstrated by extending the same chemical logic to the preparation of antibody-drug conjugate (ADC) linkers, allowing systematic evaluation of plasma stability and cytotoxic potential. By establishing a programmable capture-release chemistry for the synthesis of enzyme-activatable molecules, this work provides a generalizable chemical foundation for the scalable and automated construction of functional small-molecule tools across biological and translational research.

biochemistry↗

bioRxiv2025

Platform of single-molecule enzyme activity-based liquid biopsy for detection of pancreatic adenocarcinoma at early stages

Liquid biopsy analyzes biomolecules in biofluids to provide critical information for the early detection of diseases and the development of more effective treatment strategies. Among these biomolecules, the functional states of proteins offer particularly valuable insights due to their direct correlation with phenotypic changes. However, current approaches for assessing protein function in liquid biopsies are often limited by low detection sensitivity. Here, we present a liquid biopsy platform that analyzes single-molecule protease/peptidase activity to detect pathological alterations in enzyme function in blood samples. The platform demonstrates potential for identifying patients with early-stage (stage I-II) pancreatic ductal adenocarcinoma (PDAC).

biochemistry↗

bioRxiv2025

Development of single-molecule enzyme activity assay for serine hydrolases using activity-based protein labeling probes

Single-molecule enzyme activity assays have proven their potential in elucidating aberrant protein function at the proteoform level. However, the limited number of targetable enzymes is the major drawback of such assays. Here, we report the development of single-molecule enzyme activity assays utilizing activity-based probes that label active enzymes in an enzyme superfamily-wide manner. A proof-of-principle using fluorophosphonate-based probes was conducted to detect the active form of serine hydrolases such as PSA and granzyme B at the single-molecule level in complex biological systems such as blood. The assay suggested the potential of active granzyme B in blood as a suitable biomarker of liver damage with specific activation of immune cells.

biochemistry↗

bioRxiv2023

Liver-specific LXR inhibition represses reverse cholesterol transport in cholesterol-fed mice

ObjectiveHigh density lipoprotein (HDL) exerts an anti-atherosclerotic effect via reverse cholesterol transport (RCT). Several phases of RCT are transcriptionally controlled by Liver X receptors (LXRs). Although macrophage LXRs reportedly promote RCT, it is still uncertain whether hepatic LXRs affect RCT in vivo. Approach and ResultsTo address this question, we induced hepatic overexpression of sulfotransferase family cytosolic 2B member 1 (Sult2b1) in mice. Sult2b1 facilitates generation of sulfated cholesterol, resulting in reduced production of LXR ligands (oxysterols), which impairs LXR signaling. Adenoviral vectors expressing Sult2b1 (Ad-Sult2b1) or luciferase were intravenously injected into mice under a normal or high-cholesterol diet. Hepatic Sult2b1 overexpression resulted in reduced expression of LXR-target genes - ATP-binding cassette transporter G5/G8, cholesterol 7 hydroxylase and LXR itself - respectively reducing or increasing cholesterol levels in HDL and apolipoprotein B-containing lipoproteins (apoB-L). A macrophage RCT assay revealed that Sult2b1 overexpression inhibited fecal excretion of macrophage-derived 3H-cholesterol only under a high-cholesterol diet. In a HDL kinetic study, Ad-Sult2b1 promoted catabolism/hepatic uptake of HDL-derived cholesterol, thereby reducing fecal excretion. We next performed an in vitro lipoprotein production assay which revealed a Sult2b1-mediated reduction/increase in HDL or apoB-L secretion from hepatocytes, respectively. Finally, in LXR/{beta} double knockout mice, hepatic Sult2b1 overexpression increased apoB-L levels, but there were no differences in HDL levels or RCT compared to the control, indicating that Sult2b1-mediated effects on HDL/RCT and apoB-L were distinct: the former was LXR-dependent, but not the latter. ConclusionsHepatic LXR inhibition negatively regulates circulating HDL levels and RCT by reducing LXR-target gene expression. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/527401v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@18ff927org.highwire.dtl.DTLVardef@12e776corg.highwire.dtl.DTLVardef@1a26ebaorg.highwire.dtl.DTLVardef@113b5c6_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2022

Unique genomic sequences in novel Mycobacterium avium subsp. hominissuis lineage enabled fine scale tracing the transmission route during pig movement

Mycobacterium avium subsp. hominissuis (MAH) is one of the most prevalent mycobacteria causing non-tuberculous mycobacterial disease in humans and animals. Of note, MAH is a major cause of mycobacterial granulomatous mesenteric lymphadenitis outbreaks in pig populations. To determine the precise source of infection of MAH in a pig farm and to clarify the epidemiological relationship among pig, human and environmental MAH lineages, we collected 50 MAH isolates from pigs reared in Japan and determined draft genome sequences of 30 isolates. A variable number of tandem repeat analysis revealed that most pig MAH isolates in Japan were closely related to North American, European and Russian human isolates but not to those from East Asian human and their residential environments. Historical recombination analysis revealed that most pig isolates could be classified into SC2/4 and SC3, which contain MAH isolated from pig, European human and environmental isolates. Half of the isolates in SC2/4 had many recombination events with MAH lineages isolated from humans in East Asia. To our surprise, four isolates belonged to a new lineage (SC5) in the global MAH population. Members of SC5 had few footprints of inter-lineage recombination in the genome, and carried 80 unique genes, most of which were located on lineage specific-genomic islands. Using unique genetic features, we were able to trace the putative transmission route via their host pigs. Together, we clarify the possibility of species-specificity of MAH in addition to local adaptation. Our results highlight two transmission routes of MAH, one exposure on pig farms from the environment and the other via pig movement. Moreover, our study also warns that the evolution of MAH in pigs is influenced by MAH from patients and their residential environments, even if the MAH are genetically distinct. HighlightsO_LIVariable number of tandem repeat analysis of Mycobacterium avium subsp. hominissuis (MAH) isolated from pigs (n=50) were conducted. C_LIO_LIDraft genome sequences of MAH (n=30) and genome analysis were conducted. C_LIO_LIPig MAHs were genetically far from East Asian human isolates and close to those of Western countries. C_LIO_LINovel MAH lineage which were transmitted farms by pig movement was found. C_LIO_LIHuman MAH isolates influenced the evolution of pig isolates. C_LI

microbiology↗

bioRxiv2021

Genomic features of Mycobacterium avium subsp. hominissuis isolated from pigs in Japan.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most important agents causing non-tuberculosis mycobacterial infection in humans and pigs. Genome analysis on MAH of human isolates has been proceeding, however, those of pigs are limited despite its potential source of infection to human. In the current study, we obtained 30 draft genome sequences of MAH of pigs reared in Japan. The 30 draft genomes consisted of 4,848,678 - 5,620,788 bp length, 4,652 - 5,388 coding genes and 46 - 75 (Med: 47) tRNAs. All isolates had restriction modification associated genes and 185 - 222 predicted virulence genes. Two isolates had tRNA arrays and one isolate had a clustered regularly interspaced short palindromic repeat (CRISPR) region. Our results will be useful for evaluation of the ecology of MAH by providing a foundation for genome-based epidemiological studies.

microbiology↗

bioRxiv2020

A pVII positioning code determines the dynamics of Adenoviral nucleoprotein structure and primes it for early gene activation

Within the virion, adenovirus DNA associates with the virus-encoded, protamine-like structural protein pVII. Whether this association is organized, and how genome packaging changes during infection and subsequent transcriptional activation is currently unknown. Here, we combined RNA-seq, MNase-seq, ChIP-seq and single genome imaging during early adenovirus infection to unveil the structure- and time-resolved dynamics of viral chromatin changes as well as their correlation with gene transcription. Our MNase mapping data indicates that the viral genome is arranged in precisely positioned nucleoprotein particles (Adenosomes), like nucleosomes. We identified 238 Adenosomes, being positioned by a DNA sequence code and protecting about 60 to 70bp of DNA. The incoming genome is more accessible at early gene loci that undergo additional chromatin de-condensation upon infection. Histone H3.3 containing nucleosomes specifically replace pVII at distinct genomic sites and at the transcription start sites of early genes. Acetylation of H3.3 is predominant at the transcription start sites, preceding transcriptional activation. Based on our results we propose a central role for the viral pVII nucleoprotein-architecture, which is required for the dynamic structural changes during early infection, including the regulation of nucleosome assembly prior to transcription initiation. Our study thus may aid the rational development of recombinant adenoviral vectors exhibiting sustained expression in gene therapy.

microbiology↗

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↗

bioRxiv2019

Larger, but not better, implicit motor adaptation ability inherent in medicated Parkinson’s disease patients: a smart-device-based study

Generating appropriate motor commands is an essential brain function. To achieve proper motor control in diverse situations, predicting future states of the environment and body and modifying the prediction are indispensable. The internal model is a promising hypothesis about brain function for generating and modifying the prediction. Although several findings support the involvement of the cerebellum in the internal model, recent results support the influence of other related brain regions on the internal model. A representative example is the motor adaptation ability in Parkinsons disease (PD) patients. Although this ability provides some hints about how dopamine deficits affect the internal model, previous findings are inconsistent; some reported a deficit in the motor adaptation ability in PD patients, but others reported that the motor adaptation ability of PD patients is comparable to that of healthy controls. A possible factor causing this inconsistency is the difference in task settings, which yield different cognitive strategies in each study. Here, we demonstrate a larger, but not better, motor adaptation ability in PD patients than healthy controls while reducing the involvement of cognitive strategies and concentrating on implicit motor adaptation abilities. This study utilizes a smart-device-based experiment that enables motor adaptation experiments anytime and anywhere with less cognitive strategy involvement. The PD patients showed a significant response to insensible environmental changes, but the response was not necessarily suitable for adapting to the changes. Our findings support compensatory or paretic cerebellar functions in PD patients from the perspective of motor adaptation.

neuroscience↗

bioRxiv2018

UPA-Seq: Prediction of Functional LncRNAs Using Differential Sensitivity to UV Crosslinking

While a large number of long noncoding RNAs (lncRNAs) are transcribed from the genome of higher eukaryotes, systematic prediction of their functionality has been challenging due to the lack of conserved sequence motifs or structures. Assuming that lncRNAs function as large ribonucleoprotein complexes and thus are easily crosslinked to proteins upon UV irradiation, we performed RNA-Seq analyses of RNAs recovered from the aqueous phase after UV irradiation and phenol-chloroform extraction (UPA-Seq). As expected, the numbers of UPA-Seq reads mapped to known functional lncRNAs were remarkably reduced upon UV irradiation. Comparison with ENCODE eCLIP data revealed that lncRNAs that exhibited greater decreases upon UV irradiation preferentially associated with proteins containing prion-like domains (PrLDs). Fluorescent in situ hybridization (FISH) analyses revealed the nuclear localization of novel functional lncRNA candidates, including one that accumulated at the site of transcription. We propose that UPA-Seq provides a useful tool for the selection of lncRNA candidates to be analyzed in depth in subsequent functional studies.

molecular biology↗