Publications by authors named "Kozo Hamada"

25 Publications

  • Page 1 of 1

A non-canonical role for pyruvate kinase M2 as a functional modulator of Ca signalling through IP receptors.

Biochim Biophys Acta Mol Cell Res 2022 Jan 11;1869(4):119206. Epub 2022 Jan 11.

Division of Hematology/Oncology, Dept. Medicine, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106, USA; Dept of Medicine, University Hospitals Cleveland Medical Center, Cleveland, OH 44106, USA; Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA. Electronic address:

Pyruvate kinase isoform M2 (PKM2) is a rate-limiting glycolytic enzyme that is widely expressed in embryonic tissues. The expression of PKM2 declines in some tissues following embryogenesis, while other pyruvate kinase isozymes are upregulated. However, PKM2 is highly expressed in cancer cells and is believed to play a role in supporting anabolic processes during tumour formation. In this study, PKM2 was identified as an inositol 1,4,5-trisphosphate receptor (IPR)-interacting protein by mass spectrometry. The PKM2:IPR interaction was further characterized by pull-down and co-immunoprecipitation assays, which showed that PKM2 interacted with all three IPR isoforms. Moreover, fluorescence microscopy indicated that both IPR and PKM2 localized at the endoplasmic reticulum. PKM2 binds to IPR at a highly conserved 21-amino acid site (corresponding to amino acids 2078-2098 in mouse type 1 IPR isoform). Synthetic peptides (denoted 'TAT-D5SD' and 'D5SD'), based on the amino acid sequence at this site, disrupted the PKM2:IPR interaction and potentiated IPR-mediated Ca release both in intact cells (TAT-D5SD peptide) and in a unidirectional Ca flux assay on permeabilized cells (D5SD peptide). The TAT-D5SD peptide did not affect the enzymatic activity of PKM2. Reducing PKM2 protein expression using siRNA increased IPR-mediated Ca signalling in intact cells without altering the ER Ca content. These data identify PKM2 as an IPR-interacting protein that inhibits intracellular Ca signalling. The elevated expression of PKM2 in cancer cells is therefore not solely connected to its canonical role in glycolytic metabolism, rather PKM2 also has a novel non-canonical role in regulating intracellular signalling.
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http://dx.doi.org/10.1016/j.bbamcr.2021.119206DOI Listing
January 2022

Bcl-xL acts as an inhibitor of IPR channels, thereby antagonizing Ca-driven apoptosis.

Cell Death Differ 2021 Nov 8. Epub 2021 Nov 8.

KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, and Leuven Kanker Instituut, Campus Gasthuisberg O/N-1 Box 802, Herestraat 49, 3000, Leuven, Belgium.

Anti-apoptotic Bcl-2-family members not only act at mitochondria but also at the endoplasmic reticulum, where they impact Ca dynamics by controlling IP receptor (IPR) function. Current models propose distinct roles for Bcl-2 vs. Bcl-xL, with Bcl-2 inhibiting IPRs and preventing pro-apoptotic Ca release and Bcl-xL sensitizing IPRs to low [IP] and promoting pro-survival Ca oscillations. We here demonstrate that Bcl-xL too inhibits IPR-mediated Ca release by interacting with the same IPR regions as Bcl-2. Via in silico superposition, we previously found that the residue K87 of Bcl-xL spatially resembled K17 of Bcl-2, a residue critical for Bcl-2's IPR-inhibitory properties. Mutagenesis of K87 in Bcl-xL impaired its binding to IPR and abrogated Bcl-xL's inhibitory effect on IPRs. Single-channel recordings demonstrate that purified Bcl-xL, but not Bcl-xL, suppressed IPR single-channel openings stimulated by sub-maximal and threshold [IP]. Moreover, we demonstrate that Bcl-xL-mediated inhibition of IPRs contributes to its anti-apoptotic properties against Ca-driven apoptosis. Staurosporine (STS) elicits long-lasting Ca elevations in wild-type but not in IPR-knockout HeLa cells, sensitizing the former to STS treatment. Overexpression of Bcl-xL in wild-type HeLa cells suppressed STS-induced Ca signals and cell death, while Bcl-xL was much less effective in doing so. In the absence of IPRs, Bcl-xL and Bcl-xL were equally effective in suppressing STS-induced cell death. Finally, we demonstrate that endogenous Bcl-xL also suppress IPR activity in MDA-MB-231 breast cancer cells, whereby Bcl-xL knockdown augmented IPR-mediated Ca release and increased the sensitivity towards STS, without altering the ER Ca content. Hence, this study challenges the current paradigm of divergent functions for Bcl-2 and Bcl-xL in Ca-signaling modulation and reveals that, similarly to Bcl-2, Bcl-xL inhibits IPR-mediated Ca release and IPR-driven cell death. Our work further underpins that IPR inhibition is an integral part of Bcl-xL's anti-apoptotic function.
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http://dx.doi.org/10.1038/s41418-021-00894-wDOI Listing
November 2021

Sequestration of the PKC ortholog Pck2 in stress granules as a feedback mechanism of MAPK signaling in fission yeast.

J Cell Sci 2021 01 26;134(2). Epub 2021 Jan 26.

Laboratory of Molecular Pharmacogenomics, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kindai University, Osaka 577-8502, Japan.

Protein kinase C (PKC) signaling is a highly conserved signaling module that plays a central role in a myriad of physiological processes, ranging from cell proliferation to cell death, via various signaling pathways, including MAPK signaling. Stress granules (SGs) are non-membranous cytoplasmic foci that aggregate in cells exposed to environmental stresses. Here, we explored the role of SGs in PKC/MAPK signaling activation in fission yeast. High-heat stress (HHS) induced Pmk1 MAPK activation and Pck2 translocation from the cell tips into poly(A)-binding protein (Pabp)-positive SGs. Pck2 dispersal from the cell tips required Pck2 kinase activity, and constitutively active Pck2 exhibited increased translocation to SGs. Importantly, Pmk1 deletion impaired Pck2 recruitment to SGs, indicating that MAPK activation stimulates Pck2 SG translocation. Consistently, HHS-induced SGs delayed Pck2 relocalization at the cell tips, thereby blocking subsequent Pmk1 reactivation after recovery from HHS. HHS partitioned Pck2 into the Pabp-positive SG-containing fraction, which resulted in reduced Pck2 abundance and kinase activity in the soluble fraction. Taken together, these results indicate that MAPK-dependent Pck2 SG recruitment serves as a feedback mechanism to intercept PKC/MAPK activation induced by HHS, which might underlie PKC-related diseases.
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http://dx.doi.org/10.1242/jcs.250191DOI Listing
January 2021

Type 3 Inositol 1,4,5-Trisphosphate Receptor is a Crucial Regulator of Calcium Dynamics Mediated by Endoplasmic Reticulum in HEK Cells.

Cells 2020 01 22;9(2). Epub 2020 Jan 22.

Beijing Key Laboratory of Gene Resource and Molecular Development, College of Life Sciences, Beijing Normal University, Beijing 100875, China.

Being the largest the Ca store in mammalian cells, endoplasmic reticulum (ER)-mediated Ca signalling often involves both Ca release via inositol 1, 4, 5-trisphosphate receptors (IPR) and store operated Ca entries (SOCE) through Ca release activated Ca (CRAC) channels on plasma membrane (PM). IPRs are functionally coupled with CRAC channels and other Ca handling proteins. However, it still remains less well defined as to whether IPRs could regulate ER-mediated Ca signals independent of their Ca releasing ability. To address this, we generated IPRs triple and double knockout human embryonic kidney (HEK) cell lines (IPRs-TKO, IPRs-DKO), and systemically examined ER Ca dynamics and CRAC channel activity in these cells. The results showed that the rate of ER Ca leakage and refilling, as well as SOCE were all significantly reduced in IPRs-TKO cells. And these TKO effects could be rescued by over-expression of IPR3. Further, results showed that the diminished SOCE was caused by NEDD4L-mediated ubiquitination of Orai1 protein. Together, our findings indicate that IPR3 is one crucial player in coordinating ER-mediated Ca signalling.
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http://dx.doi.org/10.3390/cells9020275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072192PMC
January 2020

IP Receptor Plasticity Underlying Diverse Functions.

Annu Rev Physiol 2020 02 15;82:151-176. Epub 2019 Nov 15.

Laboratory of Cell Calcium Signaling, Shanghai Institute for Advanced Immunochemical Studies (SIAIS), ShanghaiTech University, Shanghai, 201210, China; email:

In the body, extracellular stimuli produce inositol 1,4,5-trisphosphate (IP), an intracellular chemical signal that binds to the IP receptor (IPR) to release calcium ions (Ca) from the endoplasmic reticulum. In the past 40 years, the wide-ranging functions mediated by IPR and its genetic defects causing a variety of disorders have been unveiled. Recent cryo-electron microscopy and X-ray crystallography have resolved IPR structures and begun to integrate with concurrent functional studies, which can explicate IP-dependent opening of Ca-conducting gates placed ∼90 Å away from IP-binding sites and its regulation by Ca. This review highlights recent research progress on the IPR structure and function. We also propose how protein plasticity within IPR, which involves allosteric gating and assembly transformations accompanied by rapid and chronic structural changes, would enable it to regulate diverse functions at cellular microdomains in pathophysiological states.
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http://dx.doi.org/10.1146/annurev-physiol-021119-034433DOI Listing
February 2020

Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na, K-ATPase control of cell adhesion, proliferation, and survival.

FASEB J 2019 09 10;33(9):10193-10206. Epub 2019 Jul 10.

Department of Women's and Children's Health, Karolinska Institutet, Solna, Sweden.

The ion pump Na, K-ATPase (NKA) is a receptor for the cardiotonic steroid ouabain. Subsaturating concentration of ouabain triggers intracellular calcium oscillations, stimulates cell proliferation and adhesion, and protects from apoptosis. However, it is controversial whether ouabain-bound NKA is considered a signal transducer. To address this question, we performed a global analysis of protein phosphorylation in COS-7 cells, identifying 2580 regulated phosphorylation events on 1242 proteins upon 10- and 20-min treatment with ouabain. Regulated phosphorylated proteins include the inositol triphosphate receptor and stromal interaction molecule, which are essential for initiating calcium oscillations. Hierarchical clustering revealed that ouabain triggers a structured phosphorylation response that occurs in a well-defined, time-dependent manner and affects specific cellular processes, including cell proliferation and cell-cell junctions. We additionally identify regulation of the phosphorylation of several calcium and calmodulin-dependent protein kinases (CAMKs), including 2 sites of CAMK type II-γ (CAMK2G), a protein known to regulate apoptosis. To verify the significance of this result, CAMK2G was knocked down in primary kidney cells. CAMK2G knockdown impaired ouabain-dependent protection from apoptosis upon treatment with high glucose or serum deprivation. In conclusion, we establish NKA as the coordinator of a broad, tightly regulated phosphorylation response in cells and define CAMK2G as a downstream effector of NKA.-Panizza, E., Zhang, L., Fontana, J. M., Hamada, K., Svensson, D., Akkuratov, E. E., Scott, L., Mikoshiba, K., Brismar, H., Lehtiö, J., Aperia, A. Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na, K-ATPase control of cell adhesion, proliferation, and survival.
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http://dx.doi.org/10.1096/fj.201900445RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704450PMC
September 2019

Bcl-2 and IP compete for the ligand-binding domain of IPRs modulating Ca signaling output.

Cell Mol Life Sci 2019 Oct 16;76(19):3843-3859. Epub 2019 Apr 16.

Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, Leuven Cancer Institute (LKI), KU Leuven, Campus Gasthuisberg O/N-1 Bus 802, Herestraat 49, 3000, Leuven, Belgium.

Bcl-2 proteins have emerged as critical regulators of intracellular Ca dynamics by directly targeting and inhibiting the IP receptor (IPR), a major intracellular Ca-release channel. Here, we demonstrate that such inhibition occurs under conditions of basal, but not high IPR activity, since overexpressed and purified Bcl-2 (or its BH4 domain) can inhibit IPR function provoked by low concentration of agonist or IP, while fails to attenuate against high concentration of agonist or IP. Surprisingly, Bcl-2 remained capable of inhibiting IPR1 channels lacking the residues encompassing the previously identified Bcl-2-binding site (a.a. 1380-1408) located in the ARM2 domain, part of the modulatory region. Using a plethora of computational, biochemical and biophysical methods, we demonstrate that Bcl-2 and more particularly its BH4 domain bind to the ligand-binding domain (LBD) of IPR1. In line with this finding, the interaction between the LBD and Bcl-2 (or its BH4 domain) was sensitive to IP and adenophostin A, ligands of the IPR. Vice versa, the BH4 domain of Bcl-2 counteracted the binding of IP to the LBD. Collectively, our work reveals a novel mechanism by which Bcl-2 influences IPR activity at the level of the LBD. This allows for exquisite modulation of Bcl-2's inhibitory properties on IPRs that is tunable to the level of IP signaling in cells.
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http://dx.doi.org/10.1007/s00018-019-03091-8DOI Listing
October 2019

Ca signaling and spinocerebellar ataxia.

Biochim Biophys Acta Mol Cell Res 2018 11 16;1865(11 Pt B):1733-1744. Epub 2018 May 16.

Laboratory for Developmental Neurobiology, Brain Science Institute, Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Electronic address:

Spinocerebellar ataxia (SCA) is a neural disorder, which is caused by degenerative changes in the cerebellum. SCA is primarily characterized by gait ataxia, and additional clinical features include nystagmus, dysarthria, tremors and cerebellar atrophy. Forty-four hereditary SCAs have been identified to date, along with >35 SCA-associated genes. Despite the great diversity and distinct functionalities of the SCA-related genes, accumulating evidence supports the occurrence of a common pathophysiological event among several hereditary SCAs. Altered calcium (Ca) homeostasis in the Purkinje cells (PCs) of the cerebellum has been proposed as a possible pathological SCA trigger. In support of this, signaling events that are initiated from or lead to aberrant Ca release from the type 1 inositol 1,4,5-trisphosphate receptor (IPR1), which is highly expressed in cerebellar PCs, seem to be closely associated with the pathogenesis of several SCA types. In this review, we summarize the current research on pathological hereditary SCA events, which involve altered Ca homeostasis in PCs, through IPR1 signaling.
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http://dx.doi.org/10.1016/j.bbamcr.2018.05.009DOI Listing
November 2018

IP-mediated gating mechanism of the IP receptor revealed by mutagenesis and X-ray crystallography.

Proc Natl Acad Sci U S A 2017 05 17;114(18):4661-4666. Epub 2017 Apr 17.

Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, Saitama 351-0198, Japan;

The inositol 1,4,5-trisphosphate (IP) receptor (IPR) is an IP-gated ion channel that releases calcium ions (Ca) from the endoplasmic reticulum. The IP-binding sites in the large cytosolic domain are distant from the Ca conducting pore, and the allosteric mechanism of how IP opens the Ca channel remains elusive. Here, we identify a long-range gating mechanism uncovered by channel mutagenesis and X-ray crystallography of the large cytosolic domain of mouse type 1 IPR in the absence and presence of IP Analyses of two distinct space group crystals uncovered an IP-dependent global translocation of the curvature α-helical domain interfacing with the cytosolic and channel domains. Mutagenesis of the IPR channel revealed an essential role of a leaflet structure in the α-helical domain. These results suggest that the curvature α-helical domain relays IP-controlled global conformational dynamics to the channel through the leaflet, conferring long-range allosteric coupling from IP binding to the Ca channel.
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http://dx.doi.org/10.1073/pnas.1701420114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5422816PMC
May 2017

Aberrant calcium signaling by transglutaminase-mediated posttranslational modification of inositol 1,4,5-trisphosphate receptors.

Proc Natl Acad Sci U S A 2014 Sep 8;111(38):E3966-75. Epub 2014 Sep 8.

Laboratory for Developmental Neurobiology, Brain Science Institute, Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Calcium Oscillation Project, International Cooperative Research Project-Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan;

The inositol 1,4,5-trisphosphate receptor (IP3R) in the endoplasmic reticulum mediates calcium signaling that impinges on intracellular processes. IP3Rs are allosteric proteins comprising four subunits that form an ion channel activated by binding of IP3 at a distance. Defective allostery in IP3R is considered crucial to cellular dysfunction, but the specific mechanism remains unknown. Here we demonstrate that a pleiotropic enzyme transglutaminase type 2 targets the allosteric coupling domain of IP3R type 1 (IP3R1) and negatively regulates IP3R1-mediated calcium signaling and autophagy by locking the subunit configurations. The control point of this regulation is the covalent posttranslational modification of the Gln2746 residue that transglutaminase type 2 tethers to the adjacent subunit. Modification of Gln2746 and IP3R1 function was observed in Huntington disease models, suggesting a pathological role of this modification in the neurodegenerative disease. Our study reveals that cellular signaling is regulated by a new mode of posttranslational modification that chronically and enzymatically blocks allosteric changes in the ligand-gated channels that relate to disease states.
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http://dx.doi.org/10.1073/pnas.1409730111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4183345PMC
September 2014

Distinct roles of M1 and M3 muscarinic acetylcholine receptors controlling oscillatory and non-oscillatory [Ca2+]i increase.

Cell Calcium 2013 Aug 7;54(2):111-9. Epub 2013 Jun 7.

Department of Physiology, Juntendo University Faculty of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.

We examined ACh-induced [Ca2+]i dynamics in pancreatic acinar cells prepared from mAChR subtype-specific knockout (KO) mice. ACh did not induce any [Ca2+]i increase in the cells isolated from M1/M3 double KO mice. In the cells from M3KO mice, ACh (0.3-3 μM) caused a monotonic [Ca2+]i increase. However, we found characteristic oscillatory [Ca2+]i increases in cells from M1KO mice in lower concentrations of ACh (0.03-0.3 μM). We investigated the receptor specific pattern of [Ca2+]i increase in COS-7 cells transfected with M1 or M3 receptors. ACh induced the oscillatory [Ca2+]i increase in M3 expressing cells, but not in cells expressing M1, which exhibited monotonic [Ca2+]i increases. IP3 production detected in fluorescent indicator co-transfected cells was higher in M1 than in M3 expressing cells. From the examination of four types of M1/M3 chimera receptors we found that the carboxyl-terminal region of M3 was responsible for the generation of Ca2+ oscillations. The present results suggest that the oscillatory Ca2+ increase in response to M3 stimulation is dependent upon a moderate IP3 increase, which is suitable for causing Ca(2+)-dependent IP3-induced Ca2+ release. The C-terminal domain of M3 may contribute as a regulator of the efficiency of Gq and PLC cooperation.
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http://dx.doi.org/10.1016/j.ceca.2013.05.004DOI Listing
August 2013

A fluorescence-based assay for the measurement of S-adenosylhomocysteine hydrolase activity in biological samples.

Anal Biochem 2013 Feb 15;433(2):95-101. Epub 2012 Oct 15.

Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.

The methylation of DNA, RNA, and proteins plays crucial roles in numerous biological processes, including epigenetic control, virus replication, and cell differentiation. In mammals, the rate-limiting step of the S-adenosylmethionine-dependent methylation process is exclusively controlled by S-adenosylhomocysteine (S-AdoHcy) hydrolase (SAHH). SAHH hydrolyzes S-AdoHcy to adenosine and homocysteine (Hcy) and is therefore a potential therapeutic target for various diseases, including cancer, malaria, and viral diseases. However, a simple and highly sensitive assay for the evaluation of SAHH activity, particularly for drug discovery, had not yet been developed. Here we present the development of a fluorescence-based assay for the measurement of SAHH activity in biological samples. We combined the advantages of the detection of fluorescent thiol groups in Hcy by ThioGlo1 with the S-AdoHcy-driven enzyme-coupled reaction. Our results confirmed the reliability of the proposed assay for the measurement of the SAHH activity of purified SAHH and showed the potential of this assay for the measurement of the SAHH activity of biological samples. Therefore, the proposed SAHH activity assay may be utilized in clinical laboratories and in high-throughput screenings for the identification of new SAHH inhibitors with potentially beneficial effects on numerous pathologies.
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http://dx.doi.org/10.1016/j.ab.2012.10.014DOI Listing
February 2013

Serotonergic integration of circadian clock and ultradian sleep-wake cycles.

J Neurosci 2012 Oct;32(42):14794-803

Department of Biomedical Chemistry, Institute of International Health, The University of Tokyo Graduate School of Medicine, Bunkyo-ku, Tokyo 113-0033, Japan.

In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus generates a 24 h rhythm of sleep and arousal. While neuronal spiking activity in the SCN provides a functional circadian oscillator that propagates throughout the brain, the ultradian sleep-wake state is regulated by the basal forebrain/preoptic area (BF/POA). How this SCN circadian oscillation is integrated into the shorter sleep-wake cycles remains unclear. We examined the temporal patterns of neuronal activity in these key brain regions in freely behaving rats. Neuronal activity in various brain regions presented diurnal rhythmicity and/or sleep-wake state dependence. We identified a diurnal rhythm in the BF/POA that was selectively degraded when diurnal arousal patterns were disrupted by acute brain serotonin depletion despite robust circadian spiking activity in the SCN. Local blockade of serotonergic transmission in the BF/POA was sufficient to disrupt the diurnal sleep-wake rhythm of mice. These results suggest that the serotonergic system enables the BF/POA to couple the SCN circadian signal to ultradian sleep-wake cycles, thereby providing a potential link between circadian rhythms and psychiatric disorders.
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http://dx.doi.org/10.1523/JNEUROSCI.0793-12.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6621427PMC
October 2012

Novel biochemical manipulation of brain serotonin reveals a role of serotonin in the circadian rhythm of sleep-wake cycles.

Eur J Neurosci 2012 Jun 24;35(11):1762-70. Epub 2012 May 24.

Department of Biomedical Chemistry, Institute of International Health, The University of Tokyo Graduate School of Medicine, Tokyo, Japan.

Serotonin (5-HT) neurons have been implicated in the modulation of many physiological functions, including mood regulation, feeding, and sleep. Impaired or altered 5-HT neurotransmission appears to be involved in depression and anxiety symptoms, as well as in sleep disorders. To investigate brain 5-HT functions in sleep, we induced 5-HT deficiency through acute tryptophan depletion in rats by intraperitoneally injecting a tryptophan-degrading enzyme called tryptophan side chain oxidase I (TSOI). After the administration of TSOI (20 units), plasma tryptophan levels selectively decreased to 1-2% of those of controls within 2 h, remained under 1% for 12-24 h, and then recovered between 72 and 96 h. Following plasma tryptophan levels, brain 5-HT levels decreased to ∼30% of the control level after 6 h, remained at this low level for 20-30 h, and returned to normal after 72 h. In contrast, brain norepinephreine and dopamine levels remained unchanged. After TSOI injection, the circadian rhythms of the sleep-wake cycle and locomotive activity were lost and broken into minute(s) ultradian alternations. The hourly slow-wave sleep (SWS) time significantly increased at night, but decreased during the day, whereas rapid eye movement sleep was significantly reduced during the day. However, daily total (cumulative) SWS time was retained at the normal level. As brain 5-HT levels gradually recovered 48 h after TSOI injection, the circadian rhythms of sleep-wake cycles and locomotive activity returned to normal. Our results suggest that 5-HT with a rapid turnover rate plays an important role in the circadian rhythm of sleep-wake cycles.
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http://dx.doi.org/10.1111/j.1460-9568.2012.08077.xDOI Listing
June 2012

Revisiting channel allostery: a coherent mechanism in IP₃ and ryanodine receptors.

Sci Signal 2012 May 22;5(225):pe24. Epub 2012 May 22.

Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.

The inositol 1,4,5-trisphosphate (IP₃) receptor is an IP₃-gated calcium ion (Ca²⁺) channel that mediates intracellular IP₃-Ca²⁺ signaling. A fundamental question--how IP₃ gates the Ca²⁺ channel within the IP₃ receptor--remains unanswered. A new crystal structure of the N-terminal region of the IP₃ receptor reveals allosteric changes by ligand binding and its similarity to the corresponding region of ryanodine receptor. Docking of the crystal structures in the electron microscopy map and an IP₃ receptor-ryanodine receptor chimera consistently supported a coherent gating mechanism in these receptors. An intriguing feature was the long distance between the IP₃-binding sites and the Ca²⁺ channel, suggesting that long-range allosteric coupling occurs between these regions upon gating of the channel. These results help integrate previous knowledge on the IP₃ and ryanodine receptors and also provide a new framework for understanding the gating mechanism.
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http://dx.doi.org/10.1126/scisignal.2003148DOI Listing
May 2012

Mechanism of ER stress-induced brain damage by IP(3) receptor.

Neuron 2010 Dec;68(5):865-78

Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

Deranged Ca(2+) signaling and an accumulation of aberrant proteins cause endoplasmic reticulum (ER) stress, which is a hallmark of cell death implicated in many neurodegenerative diseases. However, the underlying mechanisms are elusive. Here, we report that dysfunction of an ER-resident Ca(2+) channel, inositol 1,4,5-trisphosphate receptor (IP(3)R), promotes cell death during ER stress. Heterozygous knockout of brain-dominant type1 IP(3)R (IP(3)R1) resulted in neuronal vulnerability to ER stress in vivo, and IP(3)R1 knockdown enhanced ER stress-induced apoptosis via mitochondria in cultured cells. The IP(3)R1 tetrameric assembly was positively regulated by the ER chaperone GRP78 in an energy-dependent manner. ER stress induced IP(3)R1 dysfunction through an impaired IP(3)R1-GRP78 interaction, which has also been observed in the brain of Huntington's disease model mice. These results suggest that IP(3)R1 senses ER stress through GRP78 to alter the Ca(2+) signal to promote neuronal cell death implicated in neurodegenerative diseases.
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http://dx.doi.org/10.1016/j.neuron.2010.11.010DOI Listing
December 2010

Potent transglutaminase inhibitors, dithio β-aminoethyl ketones.

Bioorg Med Chem Lett 2011 Jan 31;21(1):377-9. Epub 2010 Oct 31.

Laboratory for Developmental Neurobiology, Brain Development Research Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.

Potent transglutaminase inhibitors were obtained from disulfide compounds, cystamine, dimethyl cystine, and dimethyl homocystine. The disulfide bond and thiophene ring play an important role in inhibitory activity of synthesized aryl β-amino ketones.
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http://dx.doi.org/10.1016/j.bmcl.2010.10.136DOI Listing
January 2011

Potent transglutaminase inhibitors, aryl beta-aminoethyl ketones.

Bioorg Med Chem Lett 2010 Feb 22;20(3):1141-4. Epub 2009 Dec 22.

Laboratory for Developmental Neurobiology, Brain Science Institute, Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.

Aryl beta-aminoethyl ketones were discovered as potent inhibitors of tissue transglutaminase. Heteroaryl-like thiophene groups and N-benzyl N-t-butyl aminoethyl group are critical to the strong inhibitory activity of aryl beta-aminoethyl ketones.
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http://dx.doi.org/10.1016/j.bmcl.2009.12.011DOI Listing
February 2010

Visualization of inositol 1,4,5-trisphosphate receptor by atomic force microscopy.

Neurosci Lett 2006 Jan 28;391(3):102-7. Epub 2005 Sep 28.

The Division of Neural Signal Information NTT-IMSUT, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.

Inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) acts as a ligand-gated channel that mediates neuronal signals by releasing Ca(2+) from the endoplasmic reticulum. The three-dimensional (3D) structure of tetrameric IP(3)R has been demonstrated by using electron microscopy (EM) with static specimens; however, the dynamic aspects of the IP(3)R structure have never been visualized in a native environment. Here we attempt to measure the surface topography of IP(3)R in solution using atomic force microscopy (AFM). AFM revealed large protrusions extending approximately 4.3 nm above a flat membrane prepared from Spodoptera frugiperda (Sf9) cells overexpressing mouse type 1 IP(3)R (Sf9-IP(3)R1). The average diameter of the large protrusions was approximately 32 nm. A specific antibody against a cytosolic epitope close to the IP(3)-binding site enabled us to gold-label the Sf9-IP(3)R1 membrane as confirmed by EM. AFM images of the gold-labeled membrane revealed 7.7-nm high protrusions with a diameter of approximately 30 nm, which should be IP(3)R1-antibody complexes. Authentic IP(3)R1 immuno-purified from mouse cerebella had approximately the same dimensions as those of the IP(3)R-like protrusions on the membrane. Altogether, these results suggest that the large protrusions on the Sf9-IP(3)R1 membrane correspond to the cytosolic domain of IP(3)R1. Our study provides the first 3D representation of individual IP(3)R1 particles in an aqueous solution.
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http://dx.doi.org/10.1016/j.neulet.2005.08.066DOI Listing
January 2006

Enzymatic depletion of serotonin in vivo and its consequences.

Adv Exp Med Biol 2003 ;527:199-205

The Department of Biomedical Chemistry, The Institute of International Health, The University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

The serotonin (5-HT) system in the brain is a global modulator thought to tune up a unique subset of brain keynotes such as emotion, motivation, and sleep/conscious states in the deepest seats of cognition and behavior. In pursuit for coherent accounts of such higher-order issues, we have been trying to deduce the system dynamics of 5-HT in full span from the perturbation-response couples elicited by our most quick, specific, extensive, and reversible depletion of the brain 5-HT so far available via plasma precursor annihilation by injection of a tryptophan degrading enzyme (TSO) (ISTRY meetings-1986 in Cardiff, -92 in Nagoya and -98 in Hamburg). Herein discussed are the dynamics of the 5-HT depletion both in the whole brain and regional dimensions, and then the perturbation-induced manifestation of a continuous behavioral quiescence underlain by chaotic patterns of sleep/waking states. This response in sharp contrast to those by earlier serotonin depletors, prompts us to consider a serious revision of the current 5-HT scenario. In the light of our research, future directions will be discussed together with the RTD (rapid tryptophan depletion) claiming the impaired brain 5-HT turnover by a partial decline of plasma tryptophan.
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September 2004

Inositol 1,4,5-trisphosphate receptor contains multiple cavities and L-shaped ligand-binding domains.

J Mol Biol 2004 Feb;336(1):155-64

Neuroscience Research Institute and Biological Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST), Umezono 1-1-4, Tsukuba 305-8568, Japan.

Calcium concentrations are strictly regulated in all biological cells, and one of the key molecules responsible for this regulation is the inositol 1,4,5-trisphosphate receptor, which was known to form a homotetrameric Ca(2+) channel in the endoplasmic reticulum. The receptor is involved in neuronal transmission via Ca(2+) signaling and for many other functions that relate to morphological and physiological processes in living organisms. We analysed the three-dimensional structure of the ligand-free form of the receptor based on a single-particle technique using an originally developed electron microscope equipped with a helium-cooled specimen stage and an automatic particle picking system. We propose a model that explains the complex mechanism for the regulation of Ca(2+) release by co-agonists, Ca(2+), inositol 1,4,5-trisphosphate based on the structure of multiple internal cavities and a porous balloon-shaped cytoplasmic domain containing a prominent L-shaped density which was assigned by the X-ray structure of the inositol 1,4,5-trisphosphate binding domain.
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http://dx.doi.org/10.1016/j.jmb.2003.11.024DOI Listing
February 2004

Three-dimensional rearrangements within inositol 1,4,5-trisphosphate receptor by calcium.

J Biol Chem 2003 Dec 30;278(52):52881-9. Epub 2003 Oct 30.

Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.

Allosteric binding of calcium ion (Ca2+) to inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) controls channel gating within IP3R. Here, we present biochemical and electron microscopic evidence of Ca2+-sensitive structural changes in the three-dimensional structure of type 1 IP3R (IP3R1). Low concentrations of Ca2+ and high concentrations of Sr2+ and Ba2+ were shown to be effective for the limited proteolysis of IP3R1, but Mg2+ had no effect on the proteolysis. The electron microscopy and the limited proteolysis consistently demonstrated that the effective concentration of Ca2+ for conformational changes in IP3R1 was <10(-7) m and that the IP3 scarcely affected the conformational states. The structure of IP3R1 without Ca2+, as reconstructed by three-dimensional electron microscopy, had a "mushroom-like" appearance consisting of a large square-shaped head and a small channel domain linked by four thin bridges. The projection image of the "head-to-head" assembly comprising two particles confirmed the mushroom-like side view. The "windmill-like" form of IP3R1 with Ca2+ also contains the four bridges connecting from the IP3-binding domain toward the channel domain. These data suggest that the Ca2+-specific conformational change structurally regulates the IP3-triggered channel opening within IP3R1.
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http://dx.doi.org/10.1074/jbc.M309743200DOI Listing
December 2003

Carbonic anhydrase-related protein is a novel binding protein for inositol 1,4,5-trisphosphate receptor type 1.

Biochem J 2003 Jun;372(Pt 2):435-41

Division of Molecular Neurobiology, Department of Basic Medical Sciences, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Japan.

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) is an intracellular IP(3)-gated Ca(2+) channel that is located on intracellular Ca(2+) stores and modulates Ca(2+) signalling. Using the yeast two-hybrid system, we screened a mouse brain cDNA library with bait constructs for mouse IP(3)R type 1 (IP(3)R1) to identify IP(3)R1-associated proteins. In this way, we found that carbonic anhydrase-related protein (CARP) is a novel IP(3)R1-binding protein. Western blot analysis revealed that CARP is expressed exclusively in Purkinje cells of the cerebellum, in which IP(3)R1 is abundantly expressed. Immunohistochemical analysis showed that the subcellular localization of CARP in Purkinje cells is coincident with that of IP(3)R1. Biochemical analysis also showed that CARP is co-precipitated with IP(3)R1. Using deletion mutagenesis, we established that amino acids 45-291 of CARP are essential for its association with IP(3)R1, and that the CARP-binding site is located within the modulatory domain of IP(3)R1 amino acids 1387-1647. CARP inhibits IP(3) binding to IP(3)R1 by reducing the affinity of the receptor for IP(3). As reported previously, sensitivity to IP(3) for IP(3)-induced Ca(2+) release in Purkinje cells is low compared with that in other tissues. This could be due to co-expression of CARP with IP(3)R in Purkinje cells and its inhibitory effects on IP(3) binding.
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http://dx.doi.org/10.1042/BJ20030110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1223404PMC
June 2003

Two-state conformational changes in inositol 1,4,5-trisphosphate receptor regulated by calcium.

J Biol Chem 2002 Jun 29;277(24):21115-8. Epub 2002 Apr 29.

Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.

Inositol 1,4,5-trisphosphate receptor (IP3R) is a highly controlled calcium (Ca2+) channel gated by inositol 1,4,5-trisphosphate (IP3). Multiple regulators modulate IP3-triggered pore opening by binding to discrete allosteric sites within IP3R. Accordingly we have postulated that these regulators structurally control ligand gating behavior; however, no structural evidence has been available. Here we show that Ca2+, the most pivotal regulator, induced marked structural changes in the tetrameric IP3R purified from mouse cerebella. Electron microscopy of the IP3R particles revealed two distinct structures with 4-fold symmetry: a windmill structure and a square structure. Ca2+ reversibly promoted a transition from the square to the windmill with relocations of four peripheral IP3-binding domains, assigned by binding to heparin-gold. Ca2+-dependent susceptibilities to limited digestion strongly support the notion that these alterations exist. Thus, Ca2+ appeared to regulate IP3 gating activity through the rearrangement of functional domains.
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http://dx.doi.org/10.1074/jbc.C200244200DOI Listing
June 2002
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