Publications by authors named "Hidehisa Takahashi"

48 Publications

De novo ATP1A3 variants cause polymicrogyria.

Sci Adv 2021 Mar 24;7(13). Epub 2021 Mar 24.

Department of Pediatrics, Tottori Prefectural Central Hospital, Tottori 680-0901, Japan.

Polymicrogyria is a common malformation of cortical development whose etiology remains elusive. We conducted whole-exome sequencing for 124 patients with polymicrogyria and identified de novo variants in eight patients. Mutated causes functional brain diseases, including alternating hemiplegia of childhood (AHC), rapid-onset dystonia parkinsonism (RDP), and cerebellar ataxia, areflexia, pes cavus, optic nerve atrophy, and sensorineural deafness (CAPOS). However, our patients showed no clinical features of AHC, RDP, or CAPOS and had a completely different phenotype: a severe form of polymicrogyria with epilepsy and developmental delay. Detected variants had different locations in and different functional properties compared with AHC-, RDP-, or CAPOS-associated variants. In the developing cerebral cortex of mice, radial neuronal migration was impaired in neurons overexpressing the variant of the most severe patients, suggesting that this variant is involved in cortical malformation pathogenesis. We propose a previously unidentified category of polymicrogyria associated with abnormalities.
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http://dx.doi.org/10.1126/sciadv.abd2368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990330PMC
March 2021

May-Thurner Syndrome with Calcified Uterine Leiomyoma.

Intern Med 2021 Feb 15. Epub 2021 Feb 15.

Department of Cardiology, Matsudo City General Hospital, Japan.

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http://dx.doi.org/10.2169/internalmedicine.6575-20DOI Listing
February 2021

Emesis-induced facial purpura as a mask phenomenon.

BMJ Case Rep 2021 Feb 10;14(2). Epub 2021 Feb 10.

Department of Cardiology, Matsudo City General Hospital, Matsudo, Chiba, Japan.

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http://dx.doi.org/10.1136/bcr-2020-241456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878151PMC
February 2021

A case of cardiac amyloidosis in an elderly Japanese patient with amyloidogenic transthyretin Val122Ile variant.

J Cardiol Cases 2020 Nov 29;22(5):221-225. Epub 2020 Jul 29.

Department of Cardiology, Masudo City General Hospital, Matsudo City, Chiba, Japan.

A 76-year-old Japanese man with a history of stomach cancer and chronic atrial fibrillation was referred to our department with left atrial thrombus. He had a history of gastric amyloidosis diagnosed by a pathological specimen of the stomach; however, further examination for amyloidosis was not performed. The patient displayed clinical signs and symptoms of heart failure and echocardiography showed a thick left ventricular wall. Since cardiac amyloidosis was suspected, the patient underwent cardiac magnetic resonance imaging and Tc-pyrophosphate scintigraphy. These results are consistent with transthyretin amyloidosis (ATTR amyloidosis). DNA analysis of transthyretin (TTR) was performed and a heterozygous Val122Ile mutation was identified. Notably, his only son requested the analysis; however, no mutations were noted. ATTR Val122Ile is one of the mutations in TTR that are associated with hereditary amyloidosis, causing severe cardiomyopathy. The prevalence of the ATTR Val122Ile mutation is 3.9% in the African-American population. However, the occurrence of this mutation in Asian populations is very rare. This is the second reported case of the ATTR Val122Ile variant in Japan and the first case tested including familial genes. Transthyretin amyloidosis (ATTR) Val122Ile variant is rare in Asian people. This is the second case of ATTR Val122Ile variant in Japan and the first case tested including familial genes. This case suggests this mutation is present even in Asian people. It is important to evaluate transthyretin gene mutations even in elderly ATTR cardiac amyloid without apparent family history of amyloidosis. If there is a gene mutation, it is necessary to search for transthyretin mutation within the family members.>.
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http://dx.doi.org/10.1016/j.jccase.2020.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7588479PMC
November 2020

De novo variants in CELF2 that disrupt the nuclear localization signal cause developmental and epileptic encephalopathy.

Hum Mutat 2021 Jan 10;42(1):66-76. Epub 2020 Nov 10.

Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan.

We report heterozygous CELF2 (NM_006561.3) variants in five unrelated individuals: Individuals 1-4 exhibited developmental and epileptic encephalopathy (DEE) and Individual 5 had intellectual disability and autistic features. CELF2 encodes a nucleocytoplasmic shuttling RNA-binding protein that has multiple roles in RNA processing and is involved in the embryonic development of the central nervous system and heart. Whole-exome sequencing identified the following CELF2 variants: two missense variants [c.1558C>T:p.(Pro520Ser) in unrelated Individuals 1 and 2, and c.1516C>G:p.(Arg506Gly) in Individual 3], one frameshift variant in Individual 4 that removed the last amino acid of CELF2 c.1562dup:p.(Tyr521Ter), possibly resulting in escape from nonsense-mediated mRNA decay (NMD), and one canonical splice site variant, c.272-1G>C in Individual 5, also probably leading to NMD. The identified variants in Individuals 1, 2, 4, and 5 were de novo, while the variant in Individual 3 was inherited from her mosaic mother. Notably, all identified variants, except for c.272-1G>C, were clustered within 20 amino acid residues of the C-terminus, which might be a nuclear localization signal. We demonstrated the extranuclear mislocalization of mutant CELF2 protein in cells transfected with mutant CELF2 complementary DNA plasmids. Our findings indicate that CELF2 variants that disrupt its nuclear localization are associated with DEE.
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http://dx.doi.org/10.1002/humu.24130DOI Listing
January 2021

A substrate-trapping strategy to find E3 ubiquitin ligase substrates identifies Parkin and TRIM28 targets.

Commun Biol 2020 Oct 20;3(1):592. Epub 2020 Oct 20.

Department of Biochemistry, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Kita 15, Nishi 7, Kita-Ku, Sapporo, Hokkaido, 060-8638, Japan.

The identification of true substrates of an E3 ligase is biologically important but biochemically difficult. In recent years, several techniques for identifying substrates have been developed, but these approaches cannot exclude indirect ubiquitination or have other limitations. Here we develop an E3 ligase substrate-trapping strategy by fusing a tandem ubiquitin-binding entity (TUBE) with an anti-ubiquitin remnant antibody to effectively identify ubiquitinated substrates. We apply this method to one of the RBR-type ligases, Parkin, and to one of the RING-type ligases, TRIM28, and identify previously unknown substrates for TRIM28 including cyclin A2 and TFIIB. Furthermore, we find that TRIM28 promotes cyclin A2 ubiquitination and degradation at the G1/S phase and suppresses premature entry into S phase. Taken together, the results indicate that this method is a powerful tool for comprehensively identifying substrates of E3 ligases.
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http://dx.doi.org/10.1038/s42003-020-01328-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7576197PMC
October 2020

Bromodomain-containing protein 4 regulates interleukin-34 expression in mouse ovarian cancer cells.

Inflamm Regen 2020 14;40:25. Epub 2020 Oct 14.

Division of Immunobiology, Institute for Genetic Medicine, Hokkaido University, Kita-15 Nishi-7, Kita-ku, Sapporo, Hokkaido 060-0815 Japan.

Background: Interleukin (IL)-34 acts as an alternative ligand for the colony-stimulating factor-1 receptor and controls the biology of myeloid cells, including survival, proliferation, and differentiation. IL-34 has been reported to be expressed in cancer cells and to promote tumor progression and metastasis of certain cancers via the promotion of angiogenesis and immunosuppressive macrophage differentiation. We have shown in our previous reports that targeting IL-34 in chemo-resistant tumors in vitro resulted in a remarkable inhibition of tumor growth. Also, we reported poor prognosis in patients with IL-34-expressing tumor. Therefore, blocking of IL-34 is considered as a promising therapeutic strategy to suppress tumor progression. However, the molecular mechanisms that control IL-34 production are still largely unknown.

Methods: IL-34 producing ovarian cancer cell line HM-1 was treated by bromodomain and extra terminal inhibitor JQ1. The mRNA and protein expression of IL-34 was evaluated after JQ1 treatment. Chromatin immunoprecipitation was performed to confirm the involvement of bromodomain-containing protein 4 (Brd4) in the regulation of the gene. Anti-tumor effect of JQ1 was evaluated in mouse tumor model.

Results: We identified Brd4 as one of the critical molecules that regulate expression in cancer cells. Consistent with this, we found that JQ1 is capable of efficiently suppressing the recruitment of Brd4 to the promotor region of gene. Additionally, JQ1 treatment of mice bearing IL-34-producing tumor inhibited the tumor growth along with decreasing expression in the tumor.

Conclusion: The results unveiled for the first time the responsible molecule Brd4 that regulates expression in cancer cells and suggested its possibility as a treatment target.
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http://dx.doi.org/10.1186/s41232-020-00129-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556959PMC
October 2020

A phospho-switch controls RNF43-mediated degradation of Wnt receptors to suppress tumorigenesis.

Nat Commun 2020 09 15;11(1):4586. Epub 2020 Sep 15.

Department of Biochemistry, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Kita 15, Nishi 7, Kita-ku, Sapporo, Hokkaido, 060-8638, Japan.

Frequent mutation of the tumour suppressor RNF43 is observed in many cancers, particularly colon malignancies. RNF43, an E3 ubiquitin ligase, negatively regulates Wnt signalling by inducing degradation of the Wnt receptor Frizzled. In this study, we discover that RNF43 activity requires phosphorylation at a triplet of conserved serines. This phospho-regulation of RNF43 is required for zebrafish development and growth of mouse intestinal organoids. Cancer-associated mutations that abrogate RNF43 phosphorylation cooperate with active Ras to promote tumorigenesis by abolishing the inhibitory function of RNF43 in Wnt signalling while maintaining its inhibitory function in p53 signalling. Our data suggest that RNF43 mutations cooperate with KRAS mutations to promote multi-step tumorigenesis via the Wnt-Ras-p53 axis in human colon cancers. Lastly, phosphomimetic substitutions of the serine trio restored the tumour suppressive activity of extracellular oncogenic mutants. Therefore, harnessing phospho-regulation of RNF43 might be a potential therapeutic strategy for tumours with RNF43 mutations.
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http://dx.doi.org/10.1038/s41467-020-18257-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7492264PMC
September 2020

NONO Is a Negative Regulator of Promoter.

Cancer Genomics Proteomics 2020 Jul-Aug;17(4):359-367

Department of Microbiology, Faculty of Medicine, Shimane University, Shimane, Japan

Background/aim: Sex determining region Y (SRY)-box 2 (SOX2) is a transcription factor essential for the maintenance of proliferation and self-renewal of cancer stem cells and is associated with breast cancer initiation. Regulation of cancer stem cell plasticity by SOX2 requires both positive and negative SOX2 transcription factors, but the negative regulator is still largely unknown.

Materials And Methods: SOX2 promoter-binding proteins were identified by liquid chromatography-mass spectrometry/mass spectrometry, luciferase assay, and chromatin immunoprecipitation. The effects of one such transcription factor on SOX2 expression was investigated by knockdown and overexpression experiments.

Results: Non-POU domain-containing octamer-binding protein (NONO) (also known as 54-kDa nuclear RNA-binding protein, P54NRB) was identified as a SOX2 promoter-binding protein and a negative regulator of SOX2 expression. Its activity was controlled by its coiled-coil domain and the C-terminal domain.

Conclusion: These results suggest that NONO acts as a key regulator of SOX2 transcription through the repression of SOX2 promoter activity in breast cancer cells.
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http://dx.doi.org/10.21873/cgp.20195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367599PMC
February 2021

Shank2 Binds to aPKC and Controls Tight Junction Formation with Rap1 Signaling during Establishment of Epithelial Cell Polarity.

Cell Rep 2020 04;31(1):107407

Department of Molecular Biology, Yokohama City University Graduate School of Medical Science, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. Electronic address:

Epithelial cells establish apicobasal polarity by forming tight junctions (TJs) at the apical-lateral boundary, which play fundamental roles in physiological functions. An evolutionarily conserved atypical protein kinase C (aPKC)-partitioning defective (PAR) complex functions as a platform for TJ assembly during cell polarity establishment. However, how this complex converts the spatial cues into a subsequent active unit is unclear. Here, we identify an epithelial isoform of Shank2 as a mediator of the aPKC-PAR complex. Shank2 binds to and colocalizes with aPKC at apical junctional regions of polarized epithelial cells. Shank2 knockdown results in defects in TJ formation. Mechanistically, we find that the N-terminal SPN domain is required for the junctional localization of Shank2 and binds to the active form of Rap1 small GTPase, which is involved in TJ formation. Our findings suggest that a close physical and functional relationship between aPKC and Shank2-active Rap1 signaling serves as the platform for TJ assembly to regulate epithelial cell polarity.
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http://dx.doi.org/10.1016/j.celrep.2020.02.088DOI Listing
April 2020

The role of Mediator and Little Elongation Complex in transcription termination.

Nat Commun 2020 02 26;11(1):1063. Epub 2020 Feb 26.

Department of Biochemistry, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Kita 15, Nishi 7, Kita-ku, Sapporo, Hokkaido, 060-8638, Japan.

Mediator is a coregulatory complex that regulates transcription of Pol II-dependent genes. Previously, we showed that human Mediator subunit MED26 plays a role in the recruitment of Super Elongation Complex (SEC) or Little Elongation Complex (LEC) to regulate the expression of certain genes. MED26 plays a role in recruiting SEC to protein-coding genes including c-myc and LEC to small nuclear RNA (snRNA) genes. However, how MED26 engages SEC or LEC to regulate distinct genes is unclear. Here, we provide evidence that MED26 recruits LEC to modulate transcription termination of non-polyadenylated transcripts including snRNAs and mRNAs encoding replication-dependent histone (RDH) at Cajal bodies. Our findings indicate that LEC recruited by MED26 promotes efficient transcription termination by Pol II through interaction with CBC-ARS2 and NELF/DSIF, and promotes 3' end processing by enhancing recruitment of Integrator or Heat Labile Factor to snRNA or RDH genes, respectively.
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http://dx.doi.org/10.1038/s41467-020-14849-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7044329PMC
February 2020

Gain-of-Function MN1 Truncation Variants Cause a Recognizable Syndrome with Craniofacial and Brain Abnormalities.

Am J Hum Genet 2020 01 12;106(1):13-25. Epub 2019 Dec 12.

Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan. Electronic address:

MN1 was originally identified as a tumor-suppressor gene. Knockout mouse studies have suggested that Mn1 is associated with craniofacial development. However, no MN1-related phenotypes have been established in humans. Here, we report on three individuals who have de novo MN1 variants that lead to a protein lacking the carboxyl (C) terminus and who presented with severe developmental delay, craniofacial abnormalities with specific facial features, and structural abnormalities in the brain. An in vitro study revealed that the deletion of the C-terminal region led to increased protein stability, an inhibitory effect on cell proliferation, and enhanced MN1 aggregation in nuclei compared to what occurred in the wild type, suggesting that a gain-of-function mechanism is involved in this disease. Considering that C-terminal deletion increases the fraction of intrinsically disordered regions of MN1, it is possible that altered phase separation could be involved in the mechanism underlying the disease. Our data indicate that MN1 participates in transcriptional regulation of target genes through interaction with the transcription factors PBX1, PKNOX1, and ZBTB24 and that mutant MN1 impairs the binding with ZBTB24 and RING1, which is an E3 ubiquitin ligase. On the basis of our findings, we propose the model that C-terminal deletion interferes with MN1's interaction molecules related to the ubiquitin-mediated proteasome pathway, including RING1, and increases the amount of the mutant protein; this increase leads to the dysregulation of MN1 target genes by inhibiting rapid MN1 protein turnover.
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http://dx.doi.org/10.1016/j.ajhg.2019.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042485PMC
January 2020

Angiotensin II type 1 receptor-associated protein deficiency attenuates sirtuin1 expression in an immortalised human renal proximal tubule cell line.

Sci Rep 2019 11 12;9(1):16550. Epub 2019 Nov 12.

Department of Medical Science and Cardiorenal Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

The proximal tubule is a particularly important site for ageing-related kidney damage. Sirtuin 1 (SIRT1), an NAD (nicotinamide adenine dinucleotide)-dependent deacetylase in the proximal tubule, may be involved in renal injury associated with ageing. However, the mechanisms of SIRT1 regulation remain to be elucidated. We recently reported that angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP)-deficient mice displayed age-associated renal function decline and tubulointerstitial fibrosis. Our data showed that SIRT1 protein expression was reduced in ATRAP-deficient mice, although the relationship between ATRAP deficiency and age-associated renal fibrosis is still not fully understood. It is, therefore, necessary to investigate how ATRAP affects SIRT1 protein expression to resolve ageing-associated kidney dysfunction. Here, since ageing studies are inherently lengthy, we used an ex vivo model of the proximal tubule to determine the role of ATRAP in SIRT1 protein expression. We first generated a clonal immortalised human renal proximal tubule epithelial cell line (ciRPTEC) expressing AT1R and ATRAP. Using this cell line, we demonstrated that ATRAP knockdown reduced SIRT1 protein expression in the ciRPTEC but did not alter SIRT1 mRNA expression. Thus, ATRAP likely mediates SIRT1 protein abundance in ciRPTEC.
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http://dx.doi.org/10.1038/s41598-019-52566-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851135PMC
November 2019

Acquired Localized Diaphragmatic Eventration with Liver Herniation.

Intern Med 2019 07 28;58(13):1971-1972. Epub 2019 Mar 28.

Department of Cardiology, Matsudo City General Hospital, Japan.

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http://dx.doi.org/10.2169/internalmedicine.2150-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6663550PMC
July 2019

Loss of TRIM29 Alters Keratin Distribution to Promote Cell Invasion in Squamous Cell Carcinoma.

Cancer Res 2018 12 2;78(24):6795-6806. Epub 2018 Nov 2.

Department of Biochemistry, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Hokkaido, Japan.

: TRIM29 (tripartite motif-containing protein 29) is a TRIM family protein that has been implicated in breast, colorectal, and pancreatic cancers. However, its role in stratified squamous epithelial cells and tumors has not been elucidated. Here, we investigate the expression of TRIM29 in cutaneous head and neck squamous cell carcinomas (SCC) and its functions in the tumorigenesis of such cancers. TRIM29 expression was lower in malignant SCC lesions than in adjacent normal epithelial tissue or benign tumors. Lower expression of TRIM29 was associated with higher SCC invasiveness. Primary tumors of cutaneous SCC showed aberrant hypermethylation of . Depletion of TRIM29 increased cancer cell migration and invasion; conversely, overexpression of TRIM29 suppressed these. Comprehensive proteomics and immunoprecipitation analyses identified keratins and keratin-interacting protein FAM83H as TRIM29 interactors. Knockdown of TRIM29 led to ectopic keratin localization of keratinocytes. In primary tumors, lower TRIM29 expression correlated with the altered expression of keratins. Our findings reveal an unexpected role for TRIM29 in regulating the distribution of keratins, as well as in the migration and invasion of SCC. They also suggest that the TRIM29-keratin axis could serve as a diagnostic and prognostic marker in stratified epithelial tumors and may provide a target for SCC therapeutics. SIGNIFICANCE: These findings identify TRIM29 as a novel diagnostic and prognostic marker in stratified epithelial tissues.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-1495DOI Listing
December 2018

Brain-Derived Neurotrophic Factor Improves Limited Exercise Capacity in Mice With Heart Failure.

Circulation 2018 10;138(18):2064-2066

Departments of Cardiovascular Medicine (J.M., S. Takada, S.K., T.F., H.N., N.K., M.T., A.F., T.Y., T.A.), Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan.

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http://dx.doi.org/10.1161/CIRCULATIONAHA.118.035212DOI Listing
October 2018

Anti-Sez6l2 antibody detected in a patient with immune-mediated cerebellar ataxia inhibits complex formation of GluR1 and Sez6l2.

J Neurol 2018 Apr 8;265(4):962-965. Epub 2018 Feb 8.

Department of Biochemistry, Faculty of Medicine, Graduate School of Medicine, Hokkaido University, Sapporo, Hokkaido, Japan.

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http://dx.doi.org/10.1007/s00415-018-8785-zDOI Listing
April 2018

Mutations in bassoon in individuals with familial and sporadic progressive supranuclear palsy-like syndrome.

Sci Rep 2018 01 16;8(1):819. Epub 2018 Jan 16.

Department of Neurology, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan.

Clinical diagnosis of progressive supranuclear palsy (PSP) is sometimes difficult because various phenotypes have been identified. Here, we report a mutation in the bassoon (BSN) gene in a family with PSP-like syndrome. Their clinical features resembled not only those of PSP patients but also those of individuals with multiple system atrophy and Alzheimer's disease. The neuropathological findings showed a novel three + four repeat tauopathy with pallido-luysio-nigral degeneration and hippocampal sclerosis. Whole-exome analysis of this family identified a novel missense mutation in BSN. Within the pedigree, the detected BSN mutation was found only in affected individuals. Further genetic analyses were conducted in probands from four other pedigrees with PSP-like syndrome and in 41 sporadic cases. Three missense mutations in BSN that are very rarely listed in databases of healthy subjects were found in four sporadic cases. Western blot analysis of tau following the overexpression of wild-type or mutated BSN revealed the possibility that wild-type BSN reduced tau accumulation, while mutated BSN lost this function. An association between BSN and neurological diseases has not been previously reported. Our results revealed that the neurodegenerative disorder associated with the original proband's pedigree is a novel tauopathy, differing from known dementia and parkinsonism syndromes, including PSP.
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http://dx.doi.org/10.1038/s41598-018-19198-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5770378PMC
January 2018

Sez6l2 regulates phosphorylation of ADD and neuritogenesis.

Biochem Biophys Res Commun 2017 12 12;494(1-2):234-241. Epub 2017 Oct 12.

Department of Biochemistry, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Hokkaido, Japan. Electronic address:

Increasing evidence shows that immune-mediated mechanisms may contribute to the pathogenesis of central nervous system disorders including cerebellar ataxias, as indicated by the aberrant production of neuronal surface antibodies. We previously reported a patient with cerebellar ataxia associated with production of a new anti-neuronal antibody, anti-seizure-related 6 homolog like 2 (Sez6l2). Sez6l2 is a type 1 membrane protein that is highly expressed in the hippocampus and cerebellar cortex and mice lacking Sez6l2 protein family members develop ataxia. Here we used a proteomics-based approach to show that serum derived from this patient recognizes the extracellular domain of Sez6l2 and that Sez6l2 protein binds to both adducin (ADD) and glutamate receptor 1 (GluR1). Our results indicate that Sez6l2 is one of the auxiliary subunits of the AMPA receptor and acts as a scaffolding protein to link GluR1 to ADD. Furthermore, Sez6l2 overexpression upregulates ADD phosphorylation, whereas siRNA-mediated downregulation of Sez612 prevents ADD phosphorylation, suggesting that Sez6l2 modulates AMPA-ADD signal transduction.
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http://dx.doi.org/10.1016/j.bbrc.2017.10.047DOI Listing
December 2017

The novel heart-specific RING finger protein 207 is involved in energy metabolism in cardiomyocytes.

J Mol Cell Cardiol 2016 Nov 25;100:43-53. Epub 2016 Sep 25.

Department of Biochemistry, Hokkaido University Graduate School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo, Hokkaido 060-8638, Japan. Electronic address:

A failing heart shows severe energy insufficiency, and it is presumed that this energy shortage plays a critical role in the development of cardiac dysfunction. However, little is known about the mechanisms that cause energy metabolic alterations in the failing heart. Here, we show that the novel RING-finger protein 207 (RNF207), which is specifically expressed in the heart, plays a role in cardiac energy metabolism. Depletion of RNF207 in neonatal rat cardiomyocytes (NRCs) leads to a reduced cellular concentration of adenosine triphosphate (ATP) and mitochondrial dysfunction. Consistent with this result, we observed here that the expression of RNF207 was significantly reduced in mice with common cardiac diseases including heart failure. Intriguingly, proteomic approaches revealed that RNF207 interacts with the voltage-dependent anion channel (VDAC), which is considered to be a key regulator of mitochondria function, as an RNF207-interacting protein. Our findings indicate that RNF207 is involved in ATP production by cardiomyocytes, suggesting that RNF207 plays an important role in the development of heart failure.
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http://dx.doi.org/10.1016/j.yjmcc.2016.09.013DOI Listing
November 2016

p53 represses the transcription of snRNA genes by preventing the formation of little elongation complex.

Biochim Biophys Acta 2016 08 3;1859(8):975-82. Epub 2016 Jun 3.

Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido 060-8638, Japan. Electronic address:

The regulation of transcription by RNA polymerase II (Pol II) is important for a variety of cellular functions. ELL/EAF-containing little elongation complex (LEC) was found to be required for transcription of Pol II-dependent small nuclear RNA (snRNA) genes. It was shown that the tumor suppressor p53 interacts with ELL and inhibits transcription elongation activity of ELL. Here, we show that p53 inhibits interaction between ELL/EAF and ICE1 in LEC and thereby p53 represses transcription of Pol II-dependent snRNA genes through inhibiting LEC function. Furthermore, induction of p53 expression by ultraviolet (UV) irradiation decreases the occupancy of ICE1 at Pol II-dependent snRNA genes. Consistent with the results, knockdown of p53 increased both the expression of snRNA genes and the occupancy of Pol II and components of LEC at snRNA genes. Our results indicate that p53 interferes with the interaction between ELL/EAF and ICE1 and represses transcription of snRNA genes by Pol II.
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http://dx.doi.org/10.1016/j.bbagrm.2016.06.001DOI Listing
August 2016

Ribophorin II is involved in the tissue factor expression mediated by phosphatidylserine-dependent antiprothrombin antibody on monocytes.

Rheumatology (Oxford) 2016 Jun 19;55(6):1117-26. Epub 2016 Feb 19.

Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine, Sapporo.

Objective: Phosphatidylserine-dependent, also called aPS-PT, recognizes the phosphatidylserine-prothrombin complex, which is associated with APS. We have previously reported that aPS-PT induces tissue factor (TF) expression on monocytes through the p38 mitogen-activated protein kinase pathway. However, the cell surface interaction between prothrombin and aPS-PT, which is involved in the activation of cell-signalling pathways, has remained unknown. The objective of this study was to identify membrane proteins involved in the binding of prothrombin and aPS-PT to monocyte surfaces as well as the induction of TF expression.

Methods: RAW264.7 cells with FLAG-tagged prothrombin were incubated and separated using affinity chromatography with anti-FLAG antibody-conjugated Sepharose beads. Immunopurified proteins were then analysed by an online nano-liquid chromatography-tandem mass spectrometry. The binding between prothrombin and the identified protein, ribophorin II (RPN2), was analysed by ELISA and surface plasmon resonance. To elucidate the role of RPN2 in TF expression, the TF mRNA level in RAW264.7 cells treated with RPN2 small interfering RNA was determined by quantitative real-time PCR (qPCR).

Results: RPN2 was identified as a candidate molecule involved in the binding of prothrombin to the cell surface. The binding between prothrombin and RPN2 was confirmed by ELISA and surface plasmon resonance. RAW264.7 cells treated with RPN2 small interfering RNA showed significant reduction of the TF expression mediated by prothrombin and a mouse monoclonal aPS-PT.

Conclusion: We identified that RPN2 is one of the prothrombin-binding proteins on monocyte surfaces, suggesting that RPN2 is involved in the pathophysiology of thrombosis in patients with APS.
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http://dx.doi.org/10.1093/rheumatology/kew005DOI Listing
June 2016

TRIM39 negatively regulates the NFκB-mediated signaling pathway through stabilization of Cactin.

Cell Mol Life Sci 2016 Mar 12;73(5):1085-101. Epub 2015 Sep 12.

Department of Biochemistry, Hokkaido University Graduate School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo, Hokkaido, 060-8638, Japan.

NFκB is one of the central regulators of cell survival, immunity, inflammation, carcinogenesis and organogenesis. The activation of NFκB is strictly regulated by several posttranslational modifications including phosphorylation, neddylation and ubiquitination. Several types of ubiquitination play important roles in multi-step regulations of the NFκB pathway. Some of the tripartite motif-containing (TRIM) proteins functioning as E3 ubiquitin ligases are known to regulate various biological processes such as inflammatory signaling pathways. One of the TRIM family proteins, TRIM39, for which the gene has single nucleotide polymorphisms, has been identified as one of the genetic factors in Behcet's disease. However, the role of TRIM39 in inflammatory signaling had not been fully elucidated. In this study, to elucidate the function of TRIM39 in inflammatory signaling, we performed yeast two-hybrid screening using TRIM39 as a bait and identified Cactin, which has been reported to inhibit NFκB- and TLR-mediated transcriptions. We show that TRIM39 stabilizes Cactin protein and that Cactin is upregulated after TNFα stimulation. TRIM39 knockdown also causes activation of the NFκB signal. These findings suggest that TRIM39 negatively regulates the NFκB signal in collaboration with Cactin induced by inflammatory stimulants such as TNFα.
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http://dx.doi.org/10.1007/s00018-015-2040-xDOI Listing
March 2016

Chd5 Regulates MuERV-L/MERVL Expression in Mouse Embryonic Stem Cells Via H3K27me3 Modification and Histone H3.1/H3.2.

J Cell Biochem 2016 Mar 29;117(3):780-92. Epub 2015 Sep 29.

Department of Advanced Medical Initiatives, Faculty of Medicine, Kyushu University, Fukuoka 812-8582, Japan.

Chd5 is an essential factor for neuronal differentiation and spermatogenesis and is a known tumor suppressor. H3K27me3 and H3K4un are modifications recognized by Chd5; however, it remains unclear how Chd5 remodels chromatin structure. We completely disrupted the Chd5 locus using the CRISPR-Cas9 system to generate a 52 kbp long deletion and analyzed Chd5 function in mouse embryonic stem cells. Our findings show that Chd5 represses murine endogenous retrovirus-L (MuERV-L/MERVL), an endogenous retrovirus-derived retrotransposon, by regulating H3K27me3 and H3.1/H3.2 function.
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http://dx.doi.org/10.1002/jcb.25368DOI Listing
March 2016

TRIM29 regulates the assembly of DNA repair proteins into damaged chromatin.

Nat Commun 2015 Jun 22;6:7299. Epub 2015 Jun 22.

Department of Biochemistry, Hokkaido University Graduate School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo, Hokkaido 060-8638, Japan.

Although DNA double-strand break (DSB) repair is mediated by numerous proteins accumulated at DSB sites, how DNA repair proteins are assembled into damaged chromatin has not been fully elucidated. Here we show that a member of the tripartite motif protein family, TRIM29, is a histone-binding protein responsible for DNA damage response (DDR). We found that TRIM29 interacts with BRCA1-associated surveillance complex, cohesion, DNA-PKcs and components of TIP60 complex. The dynamics of the TRIM29-containing complex on H2AX nucleosomes is coordinated by a cross-talk between histone modifications. TRIM29 binds to modified histone H3 and H4 tails in the context of nucleosomes. Furthermore, chromatin binding of TRIM29 is required for the phosphorylation of H2AX and cell viability in response to ionizing radiation. Our results suggest that TRIM29 functions as a scaffold protein to assemble DNA repair proteins into chromatin followed by efficient activation of DDR.
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http://dx.doi.org/10.1038/ncomms8299DOI Listing
June 2015

TRIM29 regulates the p63-mediated pathway in cervical cancer cells.

Biochim Biophys Acta 2015 Oct 10;1853(10 Pt A):2296-305. Epub 2015 Jun 10.

Department of Biochemistry, Hokkaido University Graduate School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo, Hokkaido 060-8638, Japan. Electronic address:

Cell invasion and adhesion play an important role in cancer metastasis and are orchestrated by a complicated network of transcription factors including p63. Here, we show that a member of the tripartite motif protein family, TRIM29, is required for regulation of the p63-mediated pathway in cervical cancer cells. TRIM29 knockdown alters the adhesion and invasion activities of cervical cancer cells. TRIM29 knockdown and overexpression cause a significant decrease and increase of TAp63α expression, respectively. TRIM29 knockdown alters the expression pattern of integrins and increases ZEB1 expression. TRIM29 is required for suppression of an increase in the adhesion activity of cells by TAp63α. These findings suggest that TRIM29 regulates the p63-mediated pathway and the behavior of cervical cancer cells.
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http://dx.doi.org/10.1016/j.bbamcr.2015.05.035DOI Listing
October 2015

The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ.

Elife 2015 Apr 23;4:e05615. Epub 2015 Apr 23.

Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Japan.

Adipocyte differentiation is a strictly controlled process regulated by a series of transcriptional activators. Adipogenic signals activate early adipogenic activators and facilitate the transient formation of early enhanceosomes at target genes. These enhancer regions are subsequently inherited by late enhanceosomes. PPARγ is one of the late adipogenic activators and is known as a master regulator of adipogenesis. However, the factors that regulate PPARγ expression remain to be elucidated. Here, we show that a novel ubiquitin E3 ligase, tripartite motif protein 23 (TRIM23), stabilizes PPARγ protein and mediates atypical polyubiquitin conjugation. TRIM23 knockdown caused a marked decrease in PPARγ protein abundance during preadipocyte differentiation, resulting in a severe defect in late adipogenic differentiation, whereas it did not affect the formation of early enhanceosomes. Our results suggest that TRIM23 plays a critical role in the switching from early to late adipogenic enhanceosomes by stabilizing PPARγ protein possibly via atypical polyubiquitin conjugation.
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http://dx.doi.org/10.7554/eLife.05615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4426667PMC
April 2015

Molecular Role of RNF43 in Canonical and Noncanonical Wnt Signaling.

Mol Cell Biol 2015 Jun 30;35(11):2007-23. Epub 2015 Mar 30.

Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido, Japan

Wnt signaling pathways are tightly regulated by ubiquitination, and dysregulation of these pathways promotes tumorigenesis. It has been reported that the ubiquitin ligase RNF43 plays an important role in frizzled-dependent regulation of the Wnt/β-catenin pathway. Here, we show that RNF43 suppresses both Wnt/β-catenin signaling and noncanonical Wnt signaling by distinct mechanisms. The suppression of Wnt/β-catenin signaling requires interaction between the extracellular protease-associated (PA) domain and the cysteine-rich domain (CRD) of frizzled and the intracellular RING finger domain of RNF43. In contrast, these N-terminal domains of RNF43 are not required for inhibition of noncanonical Wnt signaling, but interaction between the C-terminal cytoplasmic region of RNF43 and the PDZ domain of dishevelled is essential for this suppression. We further show the mechanism by which missense mutations in the extracellular portion of RNF43 identified in patients with tumors activate Wnt/β-catenin signaling. Missense mutations of RNF43 change their localization from the endosome to the endoplasmic reticulum (ER), resulting in the failure of frizzled-dependent suppression of Wnt/β-catenin signaling. However, these mutants retain the ability to suppress noncanonical Wnt signaling, probably due to interaction with dishevelled. RNF43 is also one of the potential target genes of Wnt/β-catenin signaling. Our results reveal the molecular role of RNF43 and provide an insight into tumorigenesis.
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http://dx.doi.org/10.1128/MCB.00159-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4420922PMC
June 2015

MED26 regulates the transcription of snRNA genes through the recruitment of little elongation complex.

Nat Commun 2015 Jan 9;6:5941. Epub 2015 Jan 9.

Department of Biochemistry, Hokkaido University Graduate School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo, Hokkaido 060-8638, Japan.

Regulation of transcription elongation by RNA polymerase II (Pol II) is a key regulatory step in gene transcription. Recently, the little elongation complex (LEC)-which contains the transcription elongation factor ELL/EAF-was found to be required for the transcription of Pol II-dependent small nuclear RNA (snRNA) genes. Here we show that the human Mediator subunit MED26 plays a role in the recruitment of LEC to a subset of snRNA genes through direct interaction of EAF and the N-terminal domain (NTD) of MED26. Loss of MED26 in cells decreases the occupancy of LEC at a subset of snRNA genes and results in a reduction in their transcription. Our results suggest that the MED26-NTD functions as a molecular switch in the exchange of TBP-associated factor 7 (TAF7) for LEC to facilitate the transition from initiation to elongation during transcription of a subset of snRNA genes.
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http://dx.doi.org/10.1038/ncomms6941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646223PMC
January 2015