Publications by authors named "Shintaro Kira"

20 Publications

  • Page 1 of 1

Isoflurane induces Art2-Rsp5-dependent endocytosis of Bap2 in yeast.

FEBS Open Bio 2021 Sep 18. Epub 2021 Sep 18.

Center of Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Japan.

Although general anesthesia is indispensable during modern surgical procedures, the mechanism by which inhalation anesthetics act on the synaptic membrane at the molecular and cellular level is largely unknown. In this study, we used yeast cells to examine the effect of isoflurane, an inhalation anesthetic, on membrane proteins. Bap2, an amino acid transporter localized on the plasma membrane, was endocytosed when yeast cells were treated with isoflurane. Depletion of RSP5, an E3 ligase, prevented this endocytosis and Bap2 was ubiquitinated in response to isoflurane, indicating an ubiquitin-dependent process. Screening all the Rsp5 binding adaptors showed that Art2 plays a central role in this process. These results suggest that isoflurane affects Bap2 via an Art2-Rsp5-dependent ubiquitination system.
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http://dx.doi.org/10.1002/2211-5463.13302DOI Listing
September 2021

A CRISPR/Cas9-based method for seamless N-terminal protein tagging in Saccharomyces cerevisiae.

Yeast 2021 Aug 31. Epub 2021 Aug 31.

Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Japan.

Protein tagging is an effective method for characterizing a gene of interest. Tagging can be accomplished in vivo in Saccharomyces cerevisiae by chromosomal integration of a PCR-amplified cassette. However, common tagging cassettes are not suitable for in situ N-terminal tagging when we aim to preserve the gene's endogenous promoter. Existing methods require either two rounds of homologous recombination or a relatively complex cloning process to construct strains with N-terminal protein tags. Here, we describe a simple CRISPR/Cas9-based method for seamless N-terminal tagging of yeast genes that preserves their endogenous promoter. This method enables the generation of N-terminally tagged strains by introducing an expression vector containing the cas9 gene and a specific gRNA for cleaving the 5' end of the target gene's protein-coding sequence, along with donor DNA containing the tag sequence and homology arms. gRNA cloning was executed by inverse PCR instead of the conventional method. After verifying the tag, the Cas9 and gRNA expression plasmids were eliminated without using antibiotic-containing medium. By this method, we generated strains that express N-terminally tagged subunits of the TORC1 protein kinase complex and found that these strains are comparable to strains made by conventional methods. Thus, our method provides a cost-effective alternative for seamless N-terminal tagging in baker's yeast.
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http://dx.doi.org/10.1002/yea.3666DOI Listing
August 2021

Detection of fibrotic remodeling of epicardial adipose tissue in patients with atrial fibrillation: Imaging approach based on histological observation.

Heart Rhythm O2 2021 Aug 24;2(4):311-323. Epub 2021 May 24.

Department of Cardiology and Clinical Examination.

Background: Fibrotic remodeling of epicardial adipose tissue (EAT) is crucial for proinflammatory atrial myocardial fibrosis, which leads to atrial fibrillation (AF).

Objectives: We tested the hypothesis that the ratio of central to marginal adipocyte diameter in EAT represents its fibrotic remodeling. Based on a similar concept, we also tested whether the percent (%) change in EAT fat attenuation determined using computed tomographic (CT) images can detect this remodeling.

Methods: Left atrial appendages were obtained from 76 consecutive AF patients during cardiovascular surgery. EAT in the central area (central EAT: C-EAT) and that adjacent to the atrial myocardium (Marginal EAT: M-EAT) were evaluated histologically. CT images for all of the 76 patients were also analyzed.

Results: The adipocyte diameter was smaller, fibrotic remodeling of EAT (EAT fibrosis) was more severe, and infiltration of macrophages and myofibroblasts was more extensive in M-EAT than in C-EAT. EAT fibrosis was positively correlated with adipocyte diameter in C-EAT and negatively correlated in M-EAT, resulting in a positive correlation between EAT fibrosis and the ratio of central to marginal adipocyte diameter (C/M diameter ratio; r = 0.73, < .01). The C/M diameter ratio was greater in patients with persistent AF than in those with paroxysmal AF. CT images demonstrated that the %change in EAT fat attenuation was positively correlated with EAT fibrosis.

Conclusion: Our results suggest that the central-to-marginal adipocyte diameter ratio is tightly associated with fibrotic remodeling of EAT. In addition, the %change in EAT fat attenuation determined using CT imaging can detect remodeling noninvasively.
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http://dx.doi.org/10.1016/j.hroo.2021.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8369308PMC
August 2021

Vacuolar protein Tag1 and Atg1-Atg13 regulate autophagy termination during persistent starvation in .

J Cell Sci 2021 02 26;134(4). Epub 2021 Feb 26.

Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Yamadaoka 1-8, Suita, Osaka 565-0871, Japan

Under starvation conditions, cells degrade their own components via autophagy in order to provide sufficient nutrients to ensure their survival. However, even if starvation persists, the cell is not completely degraded through autophagy, implying the existence of some kind of termination mechanism. In the yeast , autophagy is terminated after 10-12 h of nitrogen starvation. In this study, we found that termination is mediated by re-phosphorylation of Atg13 by the Atg1 protein kinase, which is also affected by PP2C phosphatases, and the eventual dispersion of the pre-autophagosomal structure, also known as the phagophore assembly site (PAS). In a genetic screen, we identified an uncharacterized vacuolar membrane protein, Tag1, as a factor responsible for the termination of autophagy. Re-phosphorylation of Atg13 and eventual PAS dispersal were defective in the Δ mutant. The vacuolar luminal domain of Tag1 and autophagic progression are important for the behaviors of Tag1. Together, our findings reveal the mechanism and factors responsible for termination of autophagy in yeast.
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http://dx.doi.org/10.1242/jcs.253682DOI Listing
February 2021

A traditional herbal medicine rikkunshito prevents angiotensin II-Induced atrial fibrosis and fibrillation.

J Cardiol 2020 12 15;76(6):626-635. Epub 2020 Jul 15.

Department of Cardiology and Clinical Examination, Oita University Faculty of Medicine, 1-1 Idaigaoka, Hasama, 879-5593, Oita, Japan.

Background: Rikkunshito (RKT), a traditional herbal medicine, has been demonstrated to exert anti-inflammatory, anti-apoptotic, and anti-fibrotic effects in several organs. This study tested the hypothesis that RKT can suppress angiotensin II (AngII)-induced inflammatory atrial fibrosis and ameliorate enhanced vulnerability to atrial fibrillation (AF).

Methods: Eight-week-old male C57BL/6 mice were subcutaneously infused with either vehicle or AngII (2.0 mg/kg/day) for 2 weeks. Water or RKT at a dose of 1000 mg/kg/day were orally administered once daily for 2 weeks. Morphological, histological, and biochemical analyses were performed. AF was induced either by transesophageal burst pacing in vivo or by burst/extrastimuli in isolated perfused hearts using a Langendorff apparatus.

Results: RKT at a dose of 1000 mg/kg/day for 2 weeks attenuated atrial interstitial fibrosis and profibrotic and proinflammatory signals induced by continuous infusion of AngII. RKT attenuated AngII-induced enhanced vulnerability to AF in in vivo experiments and in isolated perfused hearts. Atractylodin, an active component of RKT, exhibited antifibrotic activity comparable to that of RKT. RKT reversed AngII-induced suppression of sirtuin 1 (Sirt1) translocation to the nuclei. RKT suppressed AngII-induced phosphorylation of IκB, overexpression of p53, and cellular apoptotic signals and apoptosis. All of the antagonizing effects of RKT against AngII were attenuated by a concomitant treatment with a growth hormone secretagogue receptor (GHSR)-inhibitor.

Conclusion: Our results demonstrated that RKT prevented atrial fibrosis and attenuated enhanced vulnerability to AF induced by AngII. The results also suggested that potentiating the GHSR-Sirt1 pathway is involved in these processes.
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http://dx.doi.org/10.1016/j.jjcc.2020.07.001DOI Listing
December 2020

Disruption of actin dynamics regulated by Rho effector mDia1 attenuates pressure overload-induced cardiac hypertrophic responses and exacerbates dysfunction.

Cardiovasc Res 2021 03;117(4):1103-1117

Department of Pharmacology, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama, Yufu, Oita 879-5593, Japan.

Aims: Cardiac hypertrophy is a compensatory response to pressure overload, leading to heart failure. Recent studies have demonstrated that Rho is immediately activated in left ventricles after pressure overload and that Rho signalling plays crucial regulatory roles in actin cytoskeleton rearrangement during cardiac hypertrophic responses. However, the mechanisms by which Rho and its downstream proteins control actin dynamics during hypertrophic responses remain not fully understood. In this study, we identified the pivotal roles of mammalian homologue of Drosophila diaphanous (mDia) 1, a Rho-effector molecule, in pressure overload-induced ventricular hypertrophy.

Methods And Results: Male wild-type (WT) and mDia1-knockout (mDia1KO) mice (10-12 weeks old) were subjected to a transverse aortic constriction (TAC) or sham operation. The heart weight/tibia length ratio, cardiomyocyte cross-sectional area, left ventricular wall thickness, and expression of hypertrophy-specific genes were significantly decreased in mDia1KO mice 3 weeks after TAC, and the mortality rate was higher at 12 weeks. Echocardiography indicated that mDia1 deletion increased the severity of heart failure 8 weeks after TAC. Importantly, we could not observe apparent defects in cardiac hypertrophic responses in mDia3-knockout mice. Microarray analysis revealed that mDia1 was involved in the induction of hypertrophy-related genes, including immediate early genes, in pressure overloaded hearts. Loss of mDia1 attenuated activation of the mechanotransduction pathway in TAC-operated mice hearts. We also found that mDia1 was involved in stretch-induced activation of the mechanotransduction pathway and gene expression of c-fos in neonatal rat ventricular cardiomyocytes (NRVMs). mDia1 regulated the filamentous/globular (F/G)-actin ratio in response to pressure overload in mice. Additionally, increases in nuclear myocardin-related transcription factors and serum response factor were perturbed in response to pressure overload in mDia1KO mice and to mechanical stretch in mDia1 depleted NRVMs.

Conclusion: mDia1, through actin dynamics, is involved in compensatory cardiac hypertrophy in response to pressure overload.
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http://dx.doi.org/10.1093/cvr/cvaa206DOI Listing
March 2021

Role of angiopoietin-like protein 2 in atrial fibrosis induced by human epicardial adipose tissue: Analysis using an organo-culture system.

Heart Rhythm 2020 09 21;17(9):1591-1601. Epub 2020 Apr 21.

Department of Cardiology and Clinical Examination, Oita University Faculty of Medicine, Oita, Japan. Electronic address:

Background: We have recently reported that peri-left atrial epicardial adipose tissue (EAT) is associated with atrial myocardial fibrosis, in which angiopoietin-like protein 2 (Angptl2) protein content in EAT is associated with atrial myocardial fibrosis.

Objective: This study aimed to examine whether Angptl2 contained in peri-left atrial EAT can induce atrial myocardial fibrosis.

Methods: Human peri-left atrial EAT and abdominal subcutaneous adipose tissue (SAT) were collected from 9 autopsy cases. EAT- or SAT-conditioned medium was dropped onto the rat left atrial epicardial surface using an organo-culture system. Conditioned medium, recombinant Angptl2, and its antibody effects on organo-cultured rat atrial myocardial fibrosis were evaluated. Angptl2 effects on cultured neonatal rat fibroblasts were also investigated.

Results: EAT-conditioned medium induced atrial fibrosis in organo-cultured rat atrium with a progressive increase in the number of myofibroblasts. The profibrotic effect of EAT was greater than that of SAT. EAT in patients with atrial fibrillation induced a more significant atrial fibrosis than in those without. Treatment with human recombinant Angptl2 induced fibrosis in organo-cultured rat atrium, which was suppressed by the concomitant treatment with Angptl2 antibody. In cultured fibroblasts, Angptl2 upregulated the expression of α-smooth muscle actin, transforming growth factor-β1, phospho-extracellular signal-regulated kinase,phospho-inhibitor of κBα, and phospho-p38 mitogen-activated protein kinase.

Conclusion: This study demonstrated that Angptl2 contained in EAT played a crucial role in EAT-induced inflammatory atrial fibrosis. The results also suggested that antagonizing the expression of Angptl2 in EAT can be a novel therapeutic approach to prevent atrial fibrillation.
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http://dx.doi.org/10.1016/j.hrthm.2020.04.027DOI Listing
September 2020

Hyponatraemia aggravates cardiac susceptibility to ischaemia/reperfusion injury.

Int J Exp Pathol 2019 10 28;100(5-6):350-358. Epub 2020 Jan 28.

Department of Cardiology and Clinical Examination, Faculty of Medicine, Oita University, Yufu city, Japan.

Hyponatraemia is defined as a serum sodium concentration of <135 mEql/L and is the most common electrolyte disturbance in patients with chronic heart failure. We hypothesize that hyponatraemia may induce Ca overload and enhance reactive oxygen species (ROS) production, which will exacerbate myocardial injury more than normonatraemia. We investigated the effect of hyponatraemia on the ability of the heart to recover from ischaemia/reperfusion episodes. Cardiomyocytes were obtained from 1- to 3-day-old Sprague Dawley rats. After isolation, cardiomyocytes were placed in Dulbecco's modified Eagle's medium (DMEM) containing low sodium concentration (110, 120, or 130 mEq/L) or normal sodium concentration (140 mEq/L) for 72 hours. Exposure of cardiomyocytes to each of the low-sodium medium significantly increased both ROS and intracellular Ca levels compared with the exposure to the normal-sodium medium. In vivo, 8-week-old male Sprague Dawley rats were divided into four groups: control group (Con), furosemide group (Fur), low-sodium diet group (Lsd) and both furosemide and low-sodium diet group (Fur + Lsd). The hearts subjected to global ischaemia exhibited considerable decrease in left ventricular developed pressure during reperfusion, and the size of infarcts induced by ischaemia/reperfusion significantly increased in the Fur, Lsd and Fur + Lsd compared with that in the Con. Hyponatraemia aggravates cardiac susceptibility to ischaemia/reperfusion injury by Ca overload and increasing in ROS levels.
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http://dx.doi.org/10.1111/iep.12338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042740PMC
October 2019

Interleukin-10 treatment attenuates sinus node dysfunction caused by streptozotocin-induced hyperglycaemia in mice.

Cardiovasc Res 2019 01;115(1):57-70

Department of Cardiology and Clinical Examination, Oita University, Faculty of Medicine, 1-1 Idaigaoka, Hasama, Oita, Japan.

Aims: Diabetes, characterized by hyperglycaemia, causes sinus node dysfunction (SND) in several rodent models. Interleukin (IL)-10, which is a potent anti-inflammatory cytokine, has been reported to decrease in obese and diabetic patients. We tested the hypothesis that administration of IL-10 inhibits the development of SND caused by hyperglycaemia in streptozotocin (STZ)-induced diabetic mice.

Methods And Results: Six-week old CL57/B6 (WT) mice were divided into the following groups: control, STZ injection, and STZ injection with systemic administration of IL-10. IL-10 knockout mice were similarly treated. STZ-induced hyperglycaemia for 8 weeks significantly depressed serum levels of IL-10, but increased several proinflammatory cytokines in WT mice. STZ-induced hyperglycaemia-reduced resting heart rate (HR), and attenuated HR response to isoproterenol in WT mice. In isolated perfused heart experiments, corrected-sinus node recovery time was prolonged in WT mice with STZ injection. Sinus node tissue isolated from the WT-STZ group showed fibrosis, abundant infiltration of macrophages, increased production of reactive oxygen species (ROS), and depressed hyperpolarization activated cyclic nucleotide-gated potassium channel 4 (HCN4). However, the changes observed in the WT-STZ group were significantly attenuated by IL-10 administration and were further exaggerated in IL-10 knockout mice. In cultured cells, preincubation of IL-10 suppressed hyperglycaemia-induced apoptotic and profibrotic signals, and overproduction of ROS. IL-10 markedly inhibited the high glucose-induced p38 activation, and activated signal transducer and activator of transcription (STAT) 3 phosphorylation.

Conclusions: Our results suggest that IL-10 attenuates ROS production, inflammation and fibrosis, and plays an important role in the inhibition of hyperglycaemia-induced SND by suppression of HCN4 downregulation. In addition, IL-10-mediated inhibition of p38 is dependent on STAT3 phosphorylation.
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http://dx.doi.org/10.1093/cvr/cvy162DOI Listing
January 2019

Association of fibrotic remodeling and cytokines/chemokines content in epicardial adipose tissue with atrial myocardial fibrosis in patients with atrial fibrillation.

Heart Rhythm 2018 11 13;15(11):1717-1727. Epub 2018 Jun 13.

Department of Cardiology and Clinical Examination, Oita University Faculty of Medicine, Oita, Japan. Electronic address:

Background: Epicardial adipose tissue (EAT) is associated with atrial fibrillation (AF), but the underlying mechanisms remain to be fully elucidated.

Objective: The purpose of this study was to examine, using human left atrial appendage (LAA) samples, the interactive relationship between the EAT profile and atrial myocardial fibrosis through histologic and biochemical analyses.

Methods: LAA samples were obtained from 59 consecutive AF patients during cardiovascular surgery. In histologic analysis, adipose tissue, atrial myocardial fibrosis, EAT fibrosis, macrophage infiltration, and matrix metalloproteinase-2 and hypoxia-inducible factor-1α (Hif-1α) expression were evaluated in LAA sections. In biochemical analysis, proinflammatory/fibrotic proteins in EAT, total collagen in left atrial (LA) myocardium, angiopoietin-like protein-2 (Angptl2)-related proteins in EAT, and proinflammatory/fibrotic proteins in serum were evaluated.

Results: Histology revealed that the severity of fibrotic remodeling of EAT was associated with LA myocardial fibrosis. Immunohistochemical and electron microscopic findings revealed that fibrotic remodeling of EAT was associated with infiltration of macrophages and myofibroblasts. Protein concentration analysis demonstrated that the total collagen in the LA myocardium was positively correlated with proinflammatory and profibrotic cytokines/chemokines, including interleukin-6, monocyte chemoattractant protein-1, tumor necrosis factor-α, vascular endothelial growth factor, and matrix metalloproteinase-2 and matrix metalloproteinase-9 in EAT. The proinflammatory and profibrotic cytokines/chemokines in EAT and the total collagen in the LA were also positively correlated with Angptl2 in EAT.

Conclusion: Our study demonstrated that fibrotic remodeling and cytokines/chemokines in peri-LA EAT were associated with atrial myocardial fibrosis as a substrate of AF. Our results also suggested that overexpression of Hif-1α and Angptl2 may be involved in these processes.
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http://dx.doi.org/10.1016/j.hrthm.2018.06.025DOI Listing
November 2018

Interleukin 10 Treatment Ameliorates High-Fat Diet-Induced Inflammatory Atrial Remodeling and Fibrillation.

Circ Arrhythm Electrophysiol 2018 05;11(5):e006040

Department of Cardiology and Clinical Examination (H.K., I.A., A.F., Y.I., S.K., T.O., S.S., T.T., K.M., K.K., M.M., M.F., S.Y., R.A., N.O., Y.N., H.A., T.S., K.A., Y.T., K.Y., M.N., N.T.).

Background: Obesity, characterized by systemic low-grade inflammation, is considered a well-known risk for atrial fibrillation. In fact, IL-10 (interleukin 10), which is a potent anti-inflammatory cytokine, has been reported to decrease in obese and diabetic patients. We tested the hypotheses forwarding that genetic deletion of IL-10 exacerbates high-fat diet (HFD)-induced obesity-caused atrial inflammation, lipidosis, fibrosis, and fibrillation and that IL-10 therapy inhibits this pathology.

Methods: Eight- to 10-week-old male CL57/B6 (wild-type) mice and IL-10 knockout mice were divided into a 12-week HFD group and a 12-week normal-fat diet (NFD) group, respectively. In addition, the effect of IL-10 administration was also investigated.

Results: HFD-induced obesity for 12 weeks significantly depressed serum levels of IL-10 but were found to increase several proinflammatory cytokines in wild-type mice. Adverse atrial remodeling, including atrial inflammation, lipidosis, and fibrosis, was induced in both wild-type and IL-10 knockout mice by HFD. Vulnerability to atrial fibrillation was also significantly enhanced by HFD. With regard to epicardial and pericardial adipose tissue, the total amount of epicardial adipose tissue+pericardial adipose tissue volume was increased by HFD. Besides, proinflammatory and profibrotic cytokines of epicardial adipose tissue+pericardial adipose tissue were also upregulated. In contrast, the protein level of adiponectin was downregulated by HFD. These HFD-induced obesity-caused adverse effects were further exaggerated in IL-10 knockout mice in comparison to wild-type mice. Systemic IL-10 administration markedly ameliorated HFD-induced obesity-caused left atrial remodeling and vulnerability to atrial fibrillation, in addition to improving the quality of epicardial adipose tissue+pericardial adipose tissue.

Conclusions: Our results highlight IL-10 treatment as a potential therapeutic approach to limit the progression of HFD-induced obesity-caused atrial fibrillation.
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http://dx.doi.org/10.1161/CIRCEP.117.006040DOI Listing
May 2018

Gtr/Ego-independent TORC1 activation is achieved through a glutamine-sensitive interaction with Pib2 on the vacuolar membrane.

PLoS Genet 2018 04 26;14(4):e1007334. Epub 2018 Apr 26.

Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.

TORC1 is a central regulator of cell growth in response to amino acids. The role of the evolutionarily conserved Gtr/Rag pathway in the regulation of TORC1 is well-established. Recent genetic studies suggest that an additional regulatory pathway, depending on the activity of Pib2, plays a role in TORC1 activation independently of the Gtr/Rag pathway. However, the interplay between the Pib2 pathway and the Gtr/Rag pathway remains unclear. In this study, we show that Pib2 and Gtr/Ego form distinct complexes with TORC1 in a mutually exclusive manner, implying dedicated functional relationships between TORC1 and Pib2 or Gtr/Rag in response to specific amino acids. Furthermore, simultaneous depletion of Pib2 and the Gtr/Ego system abolishes TORC1 activity and completely compromises the vacuolar localization of TORC1. Thus, the amino acid-dependent activation of TORC1 is achieved through the Pib2 and Gtr/Ego pathways alone. Finally, we show that glutamine induces a dose-dependent increase in Pib2-TORC1 complex formation, and that glutamine binds directly to the Pib2 complex. These data provide strong preliminary evidence for Pib2 functioning as a putative glutamine sensor in the regulation of TORC1.
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http://dx.doi.org/10.1371/journal.pgen.1007334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919408PMC
April 2018

Vacuole-mediated selective regulation of TORC1-Sch9 signaling following oxidative stress.

Mol Biol Cell 2018 02 13;29(4):510-522. Epub 2017 Dec 13.

Department of Nanobiology, Graduate School of Advanced Integration Science

Target of rapamycin complex 1 (TORC1) is a central cellular signaling coordinator that allows eukaryotic cells to adapt to the environment. In the budding yeast, , TORC1 senses nitrogen and various stressors and modulates proteosynthesis, nitrogen uptake and metabolism, stress responses, and autophagy. There is some indication that TORC1 may regulate these downstream pathways individually. However, the potential mechanisms for such differential regulation are unknown. Here we show that the serine/threonine protein kinase Sch9 branch of TORC1 signaling depends specifically on the integrity of the vacuolar membrane, and this dependency originates in changes in Sch9 localization reflected by phosphatidylinositol 3,5-bisphosphate. Moreover, oxidative stress induces the delocalization of Sch9 from vacuoles, contributing to the persistent inhibition of the Sch9 branch after stress. Thus, our results establish that regulation of the vacuolar localization of Sch9 serves as a selective switch for the Sch9 branch in divergent TORC1 signaling. We propose that the Sch9 branch integrates the intrinsic activity of TORC1 kinase and vacuolar status, which is monitored by the phospholipids of the vacuolar membrane, into the regulation of macromolecular synthesis.
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http://dx.doi.org/10.1091/mbc.E17-09-0553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6014174PMC
February 2018

Macrophage Infiltration Into the Endothelium of Atrial Tissue in Atrial Fibrillation.

Circ J 2017 10 4;81(11):1742-1744. Epub 2017 Apr 4.

Department of Cardiology and Clinical Examination, Faculty of Medicine, Oita University.

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http://dx.doi.org/10.1253/circj.CJ-16-1072DOI Listing
October 2017

Ole1, fatty acid desaturase, is required for Atg9 delivery and isolation membrane expansion during autophagy in Saccharomyces cerevisiae.

Biol Open 2017 Jan 15;6(1):35-40. Epub 2017 Jan 15.

Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829, Japan

Macroautophagy, a major degradation pathway of cytoplasmic components, is carried out through formation of a double-membrane structure, the autophagosome. Although the involvement of specific lipid species in the formation process remains largely obscure, we recently showed that mono-unsaturated fatty acids (MUFA) generated by stearoyl-CoA desaturase 1 (SCD1) are required for autophagosome formation in mammalian cells. To obtain further insight into the role of MUFA in autophagy, in this study we analyzed the autophagic phenotypes of the yeast mutant of OLE1, an orthologue of SCD1. Δole1 cells were defective in nitrogen starvation-induced autophagy, and the Cvt pathway, when oleic acid was not supplied. Defects in elongation of the isolation membrane led to a defect in autophagosome formation. In the absence of Ole1, the transmembrane protein Atg9 was not able to reach the pre-autophagosomal structure (PAS), the site of autophagosome formation. Thus, autophagosome formation requires Ole1 during the delivery of Atg9 to the PAS/autophagosome from its cellular reservoir.
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http://dx.doi.org/10.1242/bio.022053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5278431PMC
January 2017

Dynamic relocation of the TORC1-Gtr1/2-Ego1/2/3 complex is regulated by Gtr1 and Gtr2.

Mol Biol Cell 2016 Jan 25;27(2):382-96. Epub 2015 Nov 25.

Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Osaka 565-0871, Japan Graduate School of Frontier BioSciences, Osaka University, Osaka 565-0871, Japan

TORC1 regulates cellular growth, metabolism, and autophagy by integrating various signals, including nutrient availability, through the small GTPases RagA/B/C/D in mammals and Gtr1/2 in budding yeast. Rag/Gtr is anchored to the lysosomal/vacuolar membrane by the scaffold protein complex Ragulator/Ego. Here we show that Ego consists of Ego1 and Ego3, and novel subunit Ego2. The ∆ego2 mutant exhibited only partial defects both in Gtr1-dependent TORC1 activation and Gtr1 localization on the vacuole. Ego1/2/3, Gtr1/2, and Tor1/Tco89 were colocalized on the vacuole and associated puncta. When Gtr1 was in its GTP-bound form and TORC1 was active, these proteins were preferentially localized on the vacuolar membrane, whereas when Gtr1 was in its GDP-bound form, they were mostly localized on the puncta. The localization of TORC1 to puncta was further facilitated by direct binding to Gtr2, which is involved in suppression of TORC1 activity. Thus regulation of TORC1 activity through Gtr1/Gtr2 is tightly coupled to the dynamic relocation of these proteins.
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http://dx.doi.org/10.1091/mbc.E15-07-0470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4713139PMC
January 2016

Reciprocal conversion of Gtr1 and Gtr2 nucleotide-binding states by Npr2-Npr3 inactivates TORC1 and induces autophagy.

Autophagy 2014 Sep 30;10(9):1565-78. Epub 2014 Jun 30.

Center for Frontier Oral Science; Graduate School of Dentistry; Osaka University, Osaka, Japan; Graduate School of Frontier Bioscience; Osaka University; Osaka, Japan.

Autophagy is an intracellular degradation process that delivers cytosolic material to lysosomes and vacuoles. To investigate the mechanisms that regulate autophagy, we performed a genome-wide screen using a yeast deletion-mutant collection, and found that Npr2 and Npr3 mutants were defective in autophagy. Their mammalian homologs, NPRL2 and NPRL3, were also involved in regulation of autophagy. Npr2-Npr3 function upstream of Gtr1-Gtr2, homologs of the mammalian RRAG GTPase complex, which is crucial for TORC1 regulation. Both npr2∆ mutants and a GTP-bound Gtr1 mutant suppressed autophagy and increased Tor1 vacuole localization. Furthermore, Gtr2 binds to the TORC1 subunit Kog1. A GDP-bound Gtr1 mutant induced autophagy even under nutrient-rich conditions, and this effect was dependent on the direct binding of Gtr2 to Kog1. These results revealed that 2 molecular mechanisms, Npr2-Npr3-dependent GTP hydrolysis of Gtr1 and direct binding of Gtr2 to Kog1, are involved in TORC1 inactivation and autophagic induction.
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http://dx.doi.org/10.4161/auto.29397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4206535PMC
September 2014

The transcription factors Atf1 and Pcr1 are essential for transcriptional induction of the extracellular maltase Agl1 in fission yeast.

PLoS One 2013 5;8(11):e80572. Epub 2013 Nov 5.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Japan ; Department of Biochemistry, Shimane University School of Medicine, Izumo, Japan ; PRESTO, Japan Science and Technology Agency (JST), Saitama, Japan.

The fission yeast Schizosaccharomyces pombe secretes the extracellular maltase Agl1, which hydrolyzes maltose into glucose, thereby utilizing maltose as a carbon source. Whether other maltases contribute to efficient utilization of maltose and how Agl1 expression is regulated in response to switching of carbon sources are unknown. In this study, we show that three other possible maltases and the maltose transporter Sut1 are not required for efficient utilization of maltose. Transcription of agl1 was induced when the carbon source was changed from glucose to maltose. This was dependent on Atf1 and Pcr1, which are highly conserved transcription factors that regulate stress-responsive genes in various stress conditions. Atf1 and Pcr1 generally bind the TGACGT motif as a heterodimer. The agl1 gene lacks the exact motif, but has many degenerate TGACGT motifs in its promoter and coding region. When the carbon source was switched from glucose to maltose, Atf1 and Pcr1 associated with the promoters and coding regions of agl1, fbp1, and gpx1, indicating that the Atf1-Pcr1 heteromer binds a variety of regions in its target genes to induce their transcription. In addition, the association of Mediator with these genes was dependent on Atf1 and Pcr1. These data indicate that Atf1 and Pcr1 induce the transcription of agl1, which allows efficient utilization of extracellular maltose.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080572PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3818258PMC
June 2014

TRAPPIII is responsible for vesicular transport from early endosomes to Golgi, facilitating Atg9 cycling in autophagy.

J Cell Sci 2013 Nov 28;126(Pt 21):4963-73. Epub 2013 Aug 28.

Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Osaka University, 565-0871 Osaka, Japan.

Autophagy is a bulk protein-degradation process that is regulated by many factors. In this study, we quantitatively assessed the contribution of each essential yeast gene to autophagy. Of the contributing factors that we identified, we focused on the TRAPPIII complex, which was recently shown to act as a guanine-nucleotide exchange factor for the Rab small GTPase Ypt1. Autophagy is defective in the TRAPPIII mutant under nutrient-rich conditions (Cvt pathway), but starvation-induced autophagy is only partially affected. Here, we show that TRAPPIII functions at the Golgi complex to receive general retrograde vesicle traffic from early endosomes. Cargo proteins in this TRAPPIII-dependent pathway include Atg9, a transmembrane protein that is essential for autophagy, and Snc1, a SNARE unrelated to autophagy. When cells were starved, further disruption of vesicle movement from late endosomes to the Golgi caused defects in Atg9 trafficking and autophagy. Thus, TRAPPIII-dependent sorting pathways provide Atg9 reservoirs for pre-autophagosomal structure and phagophore assembly sites under nutrient-rich conditions, whereas the late endosome-to-Golgi pathway is added to these reservoirs when nutrients are limited. This clarification of the role of TRAPPIII elucidates how general membrane traffic contributes to autophagy.
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http://dx.doi.org/10.1242/jcs.131318DOI Listing
November 2013
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