Biology subjects

Sugiyama, T.

Publications and source records attributed to Sugiyama, T..

9 recordsLinked to original sources

bioRxiv2026

Discovery of dihydroxy-enone-type protein-bound ceramides as the dominant type in human stratum corneum

Protein-bound ceramides are a specialized subclass of ceramides that are essential for skin barrier function, and their defective formation leads to severe skin disorder ichthyosis. Despite their biological importance, the precise molecular structures of protein-bound ceramides have remained incompletely defined, largely due to the technical challenges arising from their unique covalent linkage between lipid and protein components with highly distinct physicochemical properties. Using mass-spectrometry-based analyses of acylceramide moieties derived from protein-bound ceramides, we investigated whether epoxy-enone (EE)-type protein-bound ceramides present in mouse epidermis are conserved in human skin. Although EE-type protein-bound ceramides were detectable in the human stratum corneum, they constituted only a minor fraction of the ceramides in this tissue. Instead, we discovered a previously unrecognized class of protein-bound ceramides, termed dihydroxy-enone (DE)-type protein-bound ceramides, as the predominant class in human skin. These DE-type ceramides are generated through hydrolytic opening of the epoxide moiety of EE-type ceramides. In contrast, DE-type protein-bound ceramides were present in mouse epidermis at much lower levels. DE-type acylceramides appeared as two chromatographically distinct peaks, which likely correspond to putative stereoisomers with (9R,10S) and (9R,10R) configurations. Age-dependent increases in the (9R,10S) form in mouse epidermis closely paralleled changes in the expression levels of the epoxide hydrolase Ephx3, suggesting a role for EPHX3 in the conversion of EE- to DE-type ceramides. Together, these findings further reveal molecular diversity in protein-bound ceramides and a fundamental difference between human and mouse epidermal lipid architectural organization.

biochemistry↗

bioRxiv2025

A bright synthetic near-infrared luciferin enhances the capabilities of deep-tissue bioluminescence imaging using firefly luciferases

Synthetic bioluminescence reactions exhibiting near-infrared (NIR)-shifted spectra have been explored to improve deep-tissue imaging through the design of firefly luciferin analogues. Although the NIR bioluminescence reactions improve the tissue penetration of bioluminescence signals from deep tissues, their photon output is markedly lower compared to the natural reaction with D-luciferin and firefly luciferase (Fluc), often by an order of magnitude or more. Consequently, in most instances, the sensitivity of NIR bioluminescence imaging (NIR-BLI) has not yet substantially surpassed that of BLI with the natural firefly reaction. Here, we present a synthetic firefly luciferin, named AkaSuke, that generates intense NIR bioluminescence ({lambda}max = 680 nm) in reaction with Fluc, greatly improving the detection sensitivity beyond that of the D-luciferin/Fluc reaction for targeting deep tissue. AkaSuke enables sensitive visualizations of ectopic hematogenesis through entire tissues of mice over time following transplantation of bone marrow stem cells labeled with Fluc. We additionally identify a Japanese firefly luciferase, DkumLuc1, that displays higher catalytic activities for bioluminescence emission of AkaSuke compared to typical Fluc, resulting in detection sensitivity comparable to that of AkaLumine/Akaluc reaction, one of the most sensitive bioluminescence systems for deep tissue imaging. We further propose the potential of the AkaSuke/DkumLuc1 reaction as an orthogonal pair with the AkaLumine/Akaluc for sensitive dual-target tracking in mice. Overall results suggest that AkaSuke enhances the capabilities of deep-tissue bioluminescence imaging using Fluc and its variant, and could serve as an emerging benchmark for the molecular design of NIR luciferin analogues.

cancer biology↗

bioRxiv2024

Biochemical reconstitution of a major age-related cancer mutational signature by heat-induced spontaneous deamination of 5-methylcytosine residues, repair of uracil residues, and DNA replication.

Non-enzymatic spontaneous deamination of 5-methylcytosine, producing thymine, is the proposed etiology of cancer mutational signature 1, which is the most predominant signature in all cancers. Here, the proposed mutational process was reconstituted using synthetic DNA and purified proteins. First, single-stranded DNA containing 5-methylcytosine at CpG context was incubated at an elevated temperature to accelerate spontaneous DNA damage. Then, the DNA was treated with uracil DNA glycosylase to remove uracil residues that were formed by deamination of cytosine. The resulting DNA was then used as a template for DNA synthesis by yeast DNA polymerase {delta}. The DNA products were analyzed by next-generation DNA sequencing, and mutation frequencies were quantified. The observed mutations after this process were exclusively C>T mutations at CpG context, which was very similar to signature 1. When 5-methylcytosine modification and uracil DNA glycosylase were both omitted, C>T mutations were produced on C residues in all sequence contexts, but these mutations were diminished by uracil DNA glycosylase-treatment. These results indicate that the CpG>TpG mutations were produced by the deamination of 5-methylcytosine. Additional mutations, mainly C>G, were introduced by yeast DNA polymerase {zeta} on the heat-damaged DNA, indicating that G residues of the templates were also damaged. However, the damage on G residues was not converted to mutations with DNA polymerase {delta} or {varepsilon}. These results provide biochemical evidence to support that the majority of mutations in cancers are produced by ordinary DNA replication on spontaneously damaged DNA.

molecular biology↗

bioRxiv2023

Synergistic effects of PARP inhibitors by Schlafen 11 and BRCA2-deficiency through accumulation of single-strand DNA gaps behind a fork

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis) induce synthetic lethality in breast cancer gene (BRCA)-deficient tumors. Besides the original model proposed by accumulation of double-strand DNA breaks due to the impaired homologous recombination, accumulation of single-strand DNA (ssDNA) gaps due to impaired BRCA-mediated Okazaki fragment processing has emerged as an alternative mechanism of synthetic lethality. Accordingly, PARPis induce ssDNA gaps behind a replication fork in BRCA-deficient cells. Schlafen 11 (SLFN11), a member of the SLFN family, binds replication protein A (RPA)-coated ssDNA gaps and sensitizes cancer cells to DNA-damaging anticancer agents. These facts motivated us to examine the combinational effects of SLFN11 and BRCA-deficiency on PARPis sensitivity. Here, we show that SLFN11 and BRCA2-deficiency synergistically increased sensitivity to PARPis (talazoparib, niraparib, olaparib, and veliparib) at specific concentrations, where SLFN11 alone showed marginal effects. Using chromatin-bound proteins and alkaline BrdU comet assays in human cancer cells, we revealed that ssDNA gaps induced by PARPis were increased by SLFN11 or BRCA2-deficiency and that the combination of the two had the greatest effect. SLFN11 was recruited to and colocalized with chromatin-bound RPA2 under PARPis. SLFN11 recruited around a fork under DNA damage blocked replication, whereas SLFN11 recruited behind a fork under PARPis did not. Chromatin recruitment of SLFN11 and RPA2 was attenuated by the MRE11 inhibitor mirin. Hence, our studies showed that BRCA2-deficiency increased ssDNA gaps behind a fork under PARPis treatment, where SLFN11 bound and further increased the gaps. Our findings provide a mechanistic understanding of favorable responses to PARPis in SLFN11-proficient and BRCA-deficient tumors. SignificanceThis study reveals how SLFN11 enhances the antitumor effects of PARP inhibitors in BRCA2-deficient cancer cells and highlights the importance of analyzing SLFN11 expression in addition to BRCA analysis in clinical practice.

cancer biology↗

bioRxiv2023

Development of two mouse strains conditionally expressing bright luciferases with distinct emission spectra as new tools for in vivo imaging

In vivo bioluminescence imaging (BLI) has been an invaluable noninvasive method to visualize molecular and cellular behaviors in laboratory animals. Bioluminescent reporter mice possessing luciferases for general use have been limited to a classical luciferase, Luc2, from Photinus pyralis, and have been extremely powerful for various in vivo studies. However, applicability of reporter mice for in vivo BLI could be further accelerated by increasing light intensity using other luciferases and/or improving the biodistribution of their substrates in animal body. Here, we created two Cre-dependent reporter mice incorporating luciferases: oFluc derived from Pyrocoeli matsumurai and Akaluc, both of which had been reported previously to be brighter than Luc2 when using appropriate substrates; we then tested their bioluminescence in neural tissues and other organs in living mice. When expressed throughout the body, both luciferases emitted an intense yellow (oFluc) or far-red (Akaluc) light easily visible to the naked eye. Moreover, oFluc and Akaluc were similarly bright in the pancreas for in vivo BLI. However, Akaluc was superior to oFluc for brain imaging, because its substrate, AkaLumine-HCl, was distributed to the brain more efficiently than the oFluc substrate, D-luciferin. We also demonstrated that the light produced by oFluc and Akaluc was sufficiently spectrally distinct for dual-color imaging in a single living mouse. Taken together, these novel bioluminescent reporter mice are an ideal source of cells with bright bioluminescence and may facilitate the in vivo BLI of various tissues/organs for preclinical and biomedical research in combination with a wide variety of Cre-driver mice.

bioengineering↗

bioRxiv2020

Micromanipulation of amyloplasts with optical tweezers in Arabidopsis stems

Intracellular sedimentation of highly dense, starch-filled amyloplasts toward the gravity vector is likely a key initial step for gravity sensing in plants. However, recent live-cell imaging technology revealed that most amyloplasts continuously exhibit dynamic, saltatory movements in the endodermal cells of Arabidopsis stems. These complicated movements led to questions about what type of amyloplast movement triggers gravity sensing. Here we show that a confocal microscope equipped with optical tweezers can be a powerful tool to trap and manipulate amyloplasts noninvasively, while simultaneously observing cellular responses such as vacuolar dynamics in living cells. A near-infrared ({lambda} = 1064 nm) laser that was focused into the endodermal cells at 1 mW of laser power attracted and captured amyloplasts at the laser focus. The optical force exerted on the amyloplasts was theoretically estimated to be up to 1 pN. Interestingly, endosomes and trans-Golgi networks were trapped at 30 mW but not at 1 mW, which is probably due to lower refractive indices of these organelles than that of the amyloplasts. Because amyloplasts are in close proximity to vacuolar membranes in endodermal cells, their physical interaction could be visualized in real time. The vacuolar membranes drastically stretched and deformed in response to the manipulated movements of amyloplasts by optical tweezers. Our new method provides deep insights into the biophysical properties of plant organelles in vivo and opens a new avenue for studying gravity-sensing mechanisms in plants.

plant biology↗

bioRxiv2020

Reinforcement meta-learning optimizes visuomotor learning

Reinforcement learning enables the brain to learn optimal action selection, such as go or not go, by forming state-action and action-outcome associations. Does this mechanism also optimize the brains willingness to learn, such as learn or not learn? Learning to learn by rewards, i.e., reinforcement meta-learning, is a crucial mechanism for machines to develop flexibility in learning, which is also considered in the brain without empirical examinations. Here, we show that humans learn to learn or not learn to maximize rewards in visuomotor learning tasks. We also show that this regulation of learning is not a motivational bias but is a result of an instrumental, active process, which takes into account the learning-outcome structure. Our results thus demonstrate the existence of reinforcement meta-learning in the human brain. Because motor learning is a process of minimizing sensory errors, our findings uncover an essential mechanism of interaction between reward and error.

neuroscience↗

bioRxiv2019

The Ccr4-Not complex monitors the translating ribosome for codon optimality

Control of mRNA decay rate is intimately connected to translation elongation but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. Using a combination of cryo-electron microscopy, ribosome profiling and mRNA stability assays we show recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site. This interaction only occurs when the ribosome lacks accommodated A-site tRNA, indicative of low codon optimality. Loss of Not5 results in the inability of the mRNA degradation machinery to sense codon optimality. Our analysis elucidates a physical link between the Ccr4-Not complex and the ribosome providing mechanistic insight into the coupling of decoding efficiency with mRNA stability.

molecular biology↗

bioRxiv2019

Angiocrine factors from HUVECs amplify erythroid cells

Erythropoiesis is regulated by microenvironmental factors from the vasculature. Enhanced erythropoiesis, which occurs under stress or during development, amplifies erythroid cells to meet the demand of red blood cells. This process uncouples cell division and differentiation, thus the accumulated erythroid cells remain undifferentiated in the vasculature. However, little is known about how vascular endothelial cells (ECs) regulate erythropoiesis. Here we identified that human umbilical vein endothelial cells (HUVECs) keep erythroid cells undifferentiated and amplify their number. We determined that HUVECs amplify erythroid cells via secreted angiocrine factors. The expression profile of these factors suggested that they resemble macrophage-crines for enhanced erythropoiesis. Molecularly, HUVECs mediate the activation of ERK signaling. These data indicate that angiocrine factors from HUVECs enhance erythropoiesis via the amplification of undifferentiated erythroid cells. Our study contributes to the ultimate goal of harnessing erythropoiesis to replace blood transfusions.

developmental biology↗