Biology subjects

Nakayama, Y.

Publications and source records attributed to Nakayama, Y..

7 recordsLinked to original sources

bioRxiv2026

The MscS channel from Corynebacterium glutamicum uses a non-canonical gating mechanism

The function and gating mechanism of Escherichia coli mechanosensitive channel of small conductance (EcMscS) is well understood, but it is unknown whether MscS homologs in other bacteria function the same way. Here, we show that the MscS homolog from Corynebacterium glutamicum (CgMscS) opens at a much higher membrane tension than EcMscS but has otherwise similar functional characteristics. CgMscS is also structurally similar to EcMscS but features an extended transmembrane (TM) helix 2 that forms salt bridges with the cytoplasmic cage. Compared to the closed conformation, the TM1-2 sensor paddles in the inactivated conformation are rotated but not tilted, and, unlike EcMscS, there is no change in the associated pocket lipids, thus establishing a gating mechanism distinct from EcMscS that is not based on the 'lipids-move-first' model. Because the TM2 extension is conserved in Actinobacteria but not other bacterial phyla, our findings suggest that MscS homologs have lineage-specific gating mechanisms.

biophysics↗

bioRxiv2025

Discovery of a Selective Inhibitor of ZIP14 with Therapeutic Potential for Cancer-associated Cachexia

ZIP14/SLC39A14, a membrane-bound metal transporter, is essential for systemic metal homeostasis and has been implicated in inflammatory and metabolic disorders, including cancer-associated cachexia. Despite its biological and therapeutic significance, no selective inhibitors have been identified. Here, we identify 1-phenyl-8-(2-phenylethyl)-1,3,8-triazaspiro[4.5]decan-4-one (PPTD) as the first selective small-molecule inhibitor of ZIP14. PPTD efficiently blocks ZIP14-mediated uptake of zinc, iron, manganese, and cadmium, while sparing the closely related transporter ZIP8/SLC39A8. Mechanistically, PPTD binds specifically to a pocket formed at the dimer interface of ZIP14, as revealed by AlphaFold3 structural prediction, ligand-interaction profiling, structure-activity analyses, and site-directed mutagenesis, providing direct evidence for a targeted inhibition mechanism. ZIP14-driven metal influx promotes reactive oxygen species and lipid peroxidation, leading to cytotoxicity, which PPTD effectively reverses. In vivo, PPTD ameliorates major features of cancer cachexia in mice, including weight loss, reduced survival, muscle wasting, impaired locomotor activity, and disease progression. PPTD thus provides both a chemical probe to dissect ZIP14 function and a potential therapeutic candidate for cancer cachexia, establishing a foundation for the development of therapies targeting ZIP14-mediated metal dysregulation.

biochemistry↗

bioRxiv2024

A developmental brain-wide screen identifies retrosplenial cortex as a key player in the emergence of persistent memory

Memories formed early in life are short-lived while those formed later persist. Recent work revealed that infant memories are stored in a latent state. But why they fail to be retrieved is poorly understood. Here we investigated brain-wide circuit mechanisms underlying infantile amnesia. We performed a screen that combined contextual fear conditioning, activity-dependent neuronal tagging at different postnatal ages, tissue clearing and light sheet microscopy. We observed striking developmental changes in regional activity patterns between infant, juvenile, and adult mice, including changes in the retrosplenial cortex (RSP) that aligned with the emergence of persistent memory. We then performed a series of targeted investigations of RSP structure and function across development. Chronic chemogenetic reactivation of tagged RSP ensembles during the week after learning enhanced memory in adults and juveniles, but not in infants. However, after 33 days, reactivating infant-tagged RSP ensembles recovered forgotten memories. Changes in the developmental functions of RSP memory ensembles were accompanied by changes in dendritic spine density and the likelihood that those ensembles could be reactivated by contextual cues. These studies show that RSP ensembles store latent infant memories, reveal the time course of RSP functional maturation, and suggest that immature RSP functional networks contribute to infantile amnesia.

neuroscience↗

bioRxiv2021

The first transchromosomic rat model with human chromosome 21 shows robust Down syndrome features

Progress in earlier detection and symptom management has increased life expectancy and quality of life in people with Down syndrome (DS). However, no drug has been approved to help individuals with DS live independently and fully. Although rat models could support more robust physiological, behavioral, and toxicology analysis than mouse models during preclinical validation, no DS rat model is available due to technical challenges. We developed the first transchromosomic rat model of DS, TcHSA21rat, which contains a freely segregating, EGFP-inserted, human chromosome 21 (HSA21) with >93% of its protein coding genes. RNA-Seq of neonatal forebrains demonstrates that TcHSA21rat not only expresses HSA21 genes but also has an imbalance in global gene expression. Using EGFP as a marker for trisomic cells, flow cytometry analyses of peripheral blood cells from 361 adult TcHSA21rat animals show that 81% of animals retain HSA21 in >80% of cells, the criterion for a "Down syndrome karyotype" in people. TcHSA21rat exhibits learning and memory deficits and shows increased anxiety and hyperactivity. TcHSA21rat recapitulates well-characterized DS brain morphology, including smaller brain volume and reduced cerebellar size. In addition, the rat model shows reduced cerebellar foliation, a prominent feature of DS that is not observed in DS mouse models. Moreover, TcHSA21rat exhibits anomalies in craniofacial morphology, heart development, husbandry, and stature. TcHSA21rat is a robust DS animal model that can facilitate DS basic research and provide a unique tool for preclinical validation to accelerate DS drug development.

genetics↗

bioRxiv2021

Binding mechanism underlying FIH-1 suppression caused by the N-terminal disordered region of Mint3

Mint3 is known to enhance aerobic ATP production, known as the Warburg effect, by binding to FIH-1. Since this effect is considered to be beneficial for cancer cells, the interaction is a promising target for cancer therapy. However, previous research has suggested that the interacting region of Mint3 with FIH-1 is intrinsically disordered, which makes investigation of this interaction challenging. Therefore, we adopted a physicochemical approach that combined thermodynamic studies with structural analyses in solution, to clarify the binding mechanism. First, using a combination of CD, NMR, and hydrogen/deuterium exchange mass spectrometry (HDX-MS), we confirmed that the N-terminal half, which is the interacting part of Mint3, is mostly disordered. Next, using isothermal titration calorimetry (ITC), we revealed that the interaction of Mint3 and FIH-1 produces an enormous change in enthalpy and entropy. The profile is consistent with the model that the flexibility of disordered Mint3 is drastically reduced upon binding to FIH-1. Moreover, we performed a series of ITC experiments with several types of truncated Mint3s, an effective approach since the interacting part of Mint3 is disordered, and identified 78-88 as a novel core site for binding to FIH-1. The truncation study of Mint3 also revealed the thermodynamic contribution of each part to the interaction, where one contributes to the affinity ({Delta}G), while the other only affects enthalpy ({Delta}H), by forming non-covalent bonds. This insight can serve as a foothold for further investigation of IDRs and drug development for cancer therapy.

biochemistry↗

bioRxiv2020

Erythropoietin production?by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation

The detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except PEG-bound epoetin {beta} pegol. The 22 kDa band from anemic patient urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Sever hypoxia (7% O2, 4 hr) caused a 400-fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney is the main and sole site of Epo production in control and severe hypoxia. Our method will completely change Epo doping and detection.

physiology↗

bioRxiv2019

Membrane stiffness is a key determinant of E coli MscS channel mechanosensitivity

Prokaryotic mechanosensitive (MS) channels have an intimate relationship with membrane lipids. Membrane lipids may influence channel activity by directly interacting with bacterial MS channels or by influencing the global properties of the membrane such as area stretch and bending moduli. Previous work has implicated membrane stiffness as a key determinant of the mechanosensitivity of E. coli (Ec)MscS. Here we systematically tested this hypothesis using patch fluorometry of azolectin liposomes doped with lipids of increasing area stretch moduli. Increasing DOPE content of azolectin liposomes causes a rightward shift in the tension response curve of EcMscS. These rightward shifts are further magnified by the addition of stiffer forms of PE such as the branched chain lipid DPhPE and the fully saturated lipid DSPE. Furthermore, a comparison of the branched chain lipid DPhPC to the stiffer DPhPE showed a rightward shift in the tension response curve in the presence of the stiffer DPhPE. We show that these changes are not due to changes in membrane bending rigidity as the tension threshold of EcMscS in membranes doped with PC18:1 and PC18:3 are the same, despite a two-fold difference in their bending rigidity. We also show that after prolonged pressure application sudden removal of force in softer membranes causes a rebound reactivation of EcMscS and we discuss the relevance of this phenomenon to bacterial osmoregulation. Collectively, our data demonstrate that membrane stiffness is a key determinant of the mechanosensitivity of EcMscS.

biophysics↗