19 results on '"Narkhyun Bae"'
Search Results
2. Assessing kinetics and recruitment of DNA repair factors using high content screens
- Author
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Barbara Martinez-Pastor, Giorgia G. Silveira, Thomas L. Clarke, Dudley Chung, Yuchao Gu, Claudia Cosentino, Lance S. Davidow, Gadea Mata, Sylvana Hassanieh, Jayme Salsman, Alberto Ciccia, Narkhyun Bae, Mark T. Bedford, Diego Megias, Lee L. Rubin, Alejo Efeyan, Graham Dellaire, and Raul Mostoslavsky
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Histones ,Kinetics ,Open Reading Frames ,DNA Repair Enzymes ,DNA Repair ,Humans ,Tumor Suppressor p53-Binding Protein 1 ,General Biochemistry, Genetics and Molecular Biology ,Article ,Chromatin ,DNA Damage ,High-Throughput Screening Assays - Abstract
Repair of genetic damage is coordinated in the context of chromatin, so cells dynamically modulate accessibility at DNA breaks for the recruitment of DNA damage response (DDR) factors. The identification of chromatin factors with roles in DDR has mostly relied on loss-of-function screens while lacking robust high-throughput systems to study DNA repair. In this study, we have developed two high-throughput systems that allow the study of DNA repair kinetics and the recruitment of factors to double-strand breaks in a 384-well plate format. Using a customized gain-of-function open-reading frame library ("ChromORFeome" library), we identify chromatin factors with putative roles in the DDR. Among these, we find the PHF20 factor is excluded from DNA breaks, affecting DNA repair by competing with 53BP1 recruitment. Adaptable for genetic perturbations, small-molecule screens, and large-scale analysis of DNA repair, these resources can aid our understanding and manipulation of DNA repair.
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- 2021
3. Nuclear Translocation of Glutaminase GLS2 in Human Cancer Cells Associates with Proliferation Arrest and Differentiation
- Author
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María C. Gómez-García, David Carro, Carolina Lobo, Francisco J. Alonso, Narkhyun Bae, José A. Campos-Sandoval, Raghavendra G. Mirmira, Laura Castilla, Carolina Cardona, José C. Paz, Antonia Gutierrez, Gert Lubec, Ana Peñalver, José M. Matés, Amada R. López de la Oliva, Juan A. Segura, Victoria Enrique, Mercedes Martín-Rufián, Marina García-Frutos, Javier Márquez, Fernando J. Sialana, [López de la Oliva,AR, Gómez-García,MC, Cardona,C, Castilla,L, Peñalver,A, García-Frutos,M, Carro,D, Enrique,V, Paz,JC, Alonso,FJ, Segura,JA, Matés,JM, Márquez,J] Departamento de Biología Molecular y Bioquímica, Canceromics Lab, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain and Instituto de Investigación Biomédica de Málaga (IBIMA), Málaga, Spain. [Campos-Sandoval,JA, Martín-Rufián,M, Lobo,C] Proteomics Lab, Central Facility Core, University of Málaga, Málaga, Spain. [Sialana,FJ, Lubec,G] Private Medical University of Salzburg, Salzburg, Austria. [Bae,N] Institute of Science and Technology Austria, Klosterneuburg, Austria. [Mirmira,RG] Department of Pediatrics, Indiana University School of Medicine, Indianapolis, USA. [Gutiérrez,A] Departamento de Biología Celular, Genética y Fisiología, Facultad de Ciencias, Universidad de Málaga, Instituto de Investigación Biomédica de Málaga (IBIMA). Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Málaga, Spain., and This research was financed by Grant SAF2015-64501-R from the Spanish Ministry of Economy, Industry and Competitivity (to JM and JMM).
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Proteomics ,Anatomy::Cells::Cells, Cultured::Cell Line::Cell Line, Tumor [Medical Subject Headings] ,Phenomena and Processes::Cell Physiological Phenomena::Cell Physiological Processes::Cell Differentiation [Medical Subject Headings] ,Cell cycle checkpoint ,Carcinogenesis ,Ciclo celular ,lcsh:Medicine ,medicine.disease_cause ,Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Primates::Haplorhini::Catarrhini::Hominidae::Humans [Medical Subject Headings] ,Neoplasms ,Chlorocebus aethiops ,Organisms::Eukaryota::Animals [Medical Subject Headings] ,lcsh:Science ,Mitocondrias ,Cancer ,Multidisciplinary ,Glutaminase ,Chemistry ,Cell Differentiation ,Hep G2 Cells ,Diseases::Neoplasms::Neoplastic Processes::Carcinogenesis [Medical Subject Headings] ,Diseases::Neoplasms [Medical Subject Headings] ,Cancer metabolism ,Cell biology ,Mitochondria ,Translocación genética ,COS Cells ,Anatomy::Cells::Cells, Cultured::Tumor Cells, Cultured::Cell Line, Tumor::Hep G2 Cells [Medical Subject Headings] ,Proliferación celular ,Translocation ,Cell cycle ,Phenomena and Processes::Cell Physiological Phenomena::Cell Physiological Processes::Cell Cycle::Cell Cycle Checkpoints [Medical Subject Headings] ,Article ,Downregulation and upregulation ,Cell Line, Tumor ,medicine ,Glutaminasa ,Animals ,Humans ,Author Correction ,Phenomena and Processes::Cell Physiological Phenomena::Cell Physiological Processes::Cell Growth Processes::Cell Proliferation [Medical Subject Headings] ,Cell Proliferation ,Nucleoplasm ,Anatomy::Cells::Cells, Cultured::Cell Line::Cell Line, Transformed::COS Cells [Medical Subject Headings] ,Oncogene ,Cell growth ,lcsh:R ,Cell Cycle Checkpoints ,Cancer cell ,lcsh:Q ,Chemicals and Drugs::Enzymes and Coenzymes::Enzymes::Hydrolases::Amidohydrolases::Glutaminase [Medical Subject Headings] - Abstract
Glutaminase (GA) catalyzes the first step in mitochondrial glutaminolysis playing a key role in cancer metabolic reprogramming. Humans express two types of GA isoforms: GLS and GLS2. GLS isozymes have been consistently related to cell proliferation, but the role of GLS2 in cancer remains poorly understood. GLS2 is repressed in many tumor cells and a better understanding of its function in tumorigenesis may further the development of new therapeutic approaches. We analyzed GLS2 expression in HCC, GBM and neuroblastoma cells, as well as in monkey COS-7 cells. We studied GLS2 expression after induction of differentiation with phorbol ester (PMA) and transduction with the full-length cDNA of GLS2. In parallel, we investigated cell cycle progression and levels of p53, p21 and c-Myc proteins. Using the baculovirus system, human GLS2 protein was overexpressed, purified and analyzed for posttranslational modifications employing a proteomics LC-MS/MS platform. We have demonstrated a dual targeting of GLS2 in human cancer cells. Immunocytochemistry and subcellular fractionation gave consistent results demonstrating nuclear and mitochondrial locations, with the latter being predominant. Nuclear targeting was confirmed in cancer cells overexpressing c-Myc- and GFP-tagged GLS2 proteins. We assessed the subnuclear location finding a widespread distribution of GLS2 in the nucleoplasm without clear overlapping with specific nuclear substructures. GLS2 expression and nuclear accrual notably increased by treatment of SH-SY5Y cells with PMA and it correlated with cell cycle arrest at G2/M, upregulation of tumor suppressor p53 and p21 protein. A similar response was obtained by overexpression of GLS2 in T98G glioma cells, including downregulation of oncogene c-Myc. Furthermore, human GLS2 was identified as being hypusinated by MS analysis, a posttranslational modification which may be relevant for its nuclear targeting and/or function. Our studies provide evidence for a tumor suppressor role of GLS2 in certain types of cancer. The data imply that GLS2 can be regarded as a highly mobile and multilocalizing protein translocated to both mitochondria and nuclei. Upregulation of GLS2 in cancer cells induced an antiproliferative response with cell cycle arrest at the G2/M phase.
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- 2020
4. A transcriptional coregulator, SPIN·DOC, attenuates the coactivator activity of Spindlin1
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Gianluca Sbardella, X. Mark T. Bedford, Junjie Chen, Tolkappiyan Premkumar, Narkhyun Bae, Xu Li, and Min Gao
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0301 basic medicine ,Recombinant Fusion Proteins ,Cell Cycle Proteins ,Epigenetics ,Histone methylation ,Histone modification ,Transcription repressor ,Transcriptional coactivator ,Methylation ,Biochemistry ,Histones ,03 medical and health sciences ,Histone H3 ,Transcription Factor 4 ,0302 clinical medicine ,Coactivator ,Humans ,Gene Regulation ,Protein Interaction Domains and Motifs ,Wnt Signaling Pathway ,Molecular Biology ,Transcription factor ,biology ,Chemistry ,Cell Biology ,Phosphoproteins ,Chromatin ,Cell biology ,HEK293 Cells ,030104 developmental biology ,Histone ,Gene Expression Regulation ,Gene Knockdown Techniques ,030220 oncology & carcinogenesis ,Nuclear receptor coactivator 3 ,Trans-Activators ,biology.protein ,Nuclear receptor coactivator 2 ,Carrier Proteins ,Co-Repressor Proteins ,Microtubule-Associated Proteins - Abstract
Spindlin1 (SPIN1) is a transcriptional coactivator with critical functions in embryonic development and emerging roles in cancer. SPIN1 harbors three Tudor domains, two of which engage the tail of histone H3 by reading the H3-Lys-4 trimethylation and H3-Arg-8 asymmetric dimethylation marks. To gain mechanistic insight into how SPIN1 functions as a transcriptional coactivator, here we purified its interacting proteins. We identified an uncharacterized protein (C11orf84), which we renamed SPIN1 docking protein (SPIN·DOC), that directly binds SPIN1 and strongly disrupts its histone methylation reading ability, causing it to disassociate from chromatin. The Spindlin family of coactivators has five related members (SPIN1, 2A, 2B, 3, and 4), and we found that all of them bind SPIN·DOC. It has been reported previously that SPIN1 regulates gene expression in the Wnt signaling pathway by directly interacting with transcription factor 4 (TCF4). We observed here that SPIN·DOC associates with TCF4 in a SPIN1-dependent manner and dampens SPIN1 coactivator activity in TOPflash reporter assays. Furthermore, knockdown and overexpression experiments indicated that SPIN·DOC represses the expression of a number of SPIN1-regulated genes, including those encoding ribosomal RNA and the cytokine IL1B. In conclusion, we have identified SPIN·DOC as a transcriptional repressor that binds SPIN1 and masks its ability to engage the H3-Lys-4 trimethylation activation mark.
- Published
- 2017
5. Author Correction: Nuclear Translocation of Glutaminase GLS2 in Human Cancer Cells Associates with Proliferation Arrest and Differentiation
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Marina García-Frutos, David Carro, Raghavendra G. Mirmira, Antonia Gutierrez, José A. Campos-Sandoval, María C. Gómez-García, Laura Castilla, Amada R. López de la Oliva, José M. Matés, Gert Lubec, Carolina Cardona, José C. Paz, Juan A. Segura, Carolina Lobo, Javier Márquez, Fernando J. Sialana, Narkhyun Bae, Mercedes Martín-Rufián, Ana Peñalver, Victoria Enrique, and Francisco J. Alonso
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Multidisciplinary ,Glutaminase ,Science ,Cancer research ,Medicine ,Biology ,Human cancer ,Nuclear translocation - Abstract
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
- Published
- 2021
6. MOESM6 of Histone peptide microarray screen of chromo and Tudor domains defines new histone lysine methylation interactions
- Author
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Shanle, Erin, Shinsky, Stephen, Bridgers, Joseph, Narkhyun Bae, Sagum, Cari, Krajewski, Krzysztof, Rothbart, Scott, Bedford, Mark, and Strahl, Brian
- Abstract
Additional file 6: Figure S4. CHD7 chromodomain histone peptide microarray. A) Representative array images of CHD7 chromodomain showing peptide binding indicated in red (right panel). The peptide tracer is shown in green (left panel). Positive antibody controls are outlined in white. B) Scatter plot of the relative binding of CHD7 chromodomain from two independent peptide arrays. All modified and unmodified H4 (1â 23) peptides are shown in red. All other peptides are shown in black. C) Relative binding to the indicated histone peptides from one representative array. Data were normalized to the most intense binding and the average and standard deviation of triplicate spots is shown.
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- 2017
- Full Text
- View/download PDF
7. The Molecular Background of the Differential UV Absorbance of the Human Lens in the 240-400 nm Range
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Lajos Kolozsvári, Sung Ung Kang, Akos Tiboldi, Kongzhao Li, Gert Lubec, Béla Hopp, Viktor Pajer, Narkhyun Bae, and Antal Nógrádi
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Adult ,Gel electrophoresis ,Chromatography ,medicine.diagnostic_test ,Ultraviolet Rays ,Spectrophotometry, Atomic ,General Medicine ,Middle Aged ,Biology ,Mass spectrometry ,Trypsin ,medicine.disease_cause ,Biochemistry ,Lens protein ,Electrophoresis ,medicine.anatomical_structure ,Spectrophotometry ,Lens (anatomy) ,Lens, Crystalline ,medicine ,Humans ,Electrophoresis, Gel, Two-Dimensional ,Physical and Theoretical Chemistry ,Ultraviolet ,medicine.drug - Abstract
The ultraviolet (UV) absorption of various sections of the human lens was studied and compared with protein expression paralleling differential UV absorbance in anterior and posterior lenticular tissue. The UV absorbance of serial lens cryostat sections (60 μm) and that of lens capsules was determined using a Shimadzu scanning spectrophotometer, and the absorption coefficients were calculated. Two-dimensional gel electrophoresis was performed using two pooled lenticular protein extracts (anterior and posterior sections). Protein spots were quantified and significantly different spots were identified by mass spectrometry following in-gel digestion with trypsin and chymotrypsin. The UV-C and UV-B absorption of the human lens increased toward the posterior parts of the lens. The anterior and posterior lens capsules also effectively absorbed UV radiation. Levels of molecular chaperone proteins Beta-crystallin B2 (UniProtKB ID:P43320), A3 (UniProtKB ID:P05813) and of glyceraldehyde 3-phosphate dehydrogenase (UniProtKB ID:P04406) were significantly higher in the anterior part of the lens, whereas lens proteins Beta-crystallin B1 (UniProtKB ID:P53674) and Alpha-crystallin A chain (UniProtKB ID:P02489) were higher in the posterior sections. These results provide evidence that differential UV absorption in the anterior and posterior lens is accompanied by differential protein expression.
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- 2013
8. Network of brain protein level changes in glutaminase deficient fetal mice
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Narkhyun Bae, Gert Lubec, Stephen Rayport, Lin Li, and Yvonne Wang
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Male ,Proteomics ,Heterogeneous nuclear ribonucleoprotein ,Protein subunit ,ATPase ,Biophysics ,Nerve Tissue Proteins ,Biochemistry ,Article ,Mice ,Fetus ,Glutaminase ,Tandem Mass Spectrometry ,Animals ,Electrophoresis, Gel, Two-Dimensional ,Protein kinase B ,Brain Chemistry ,Neurocalcin ,biology ,Brain ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,biology.protein ,Signal transduction ,Retinol binding ,Metabolic Networks and Pathways ,Signal Transduction - Abstract
Glutaminase is a multifunctional enzyme encoded by gene Gls involved in energy metabolism, ammonia trafficking and regeneration of neurotransmitter glutamate. To address the proteomic basis for the neurophenotypes of glutaminase-deficient mice, brain proteins from late gestation wild type, Gls +/− and Gls −/− male mice were subjected to two-dimensional gel electrophoresis, with subsequent identification by mass spectrometry using nano-LC-ESI–MS/MS. Protein spots that showed differential genotypic variation were quantified by immunoblotting. Differentially expressed proteins unambiguously identified by MS/MS included neurocalcin delta, retinol binding protein-1, reticulocalbin-3, cytoskeleton proteins fascin and tropomyosin alpha-4-chain, dihydropyrimidinase-related protein-5, apolipoprotein IV and proteins from protein metabolism proteasome subunits alpha type 2, type 7, heterogeneous nuclear ribonucleoprotein C1/C2 and H, voltage-gated anion-selective channel proteins 1 and 2, ATP synthase subunit β and transitional endoplasmic reticulum ATPase. An interaction network determined by Ingenuity Pathway Analysis revealed a link between glutaminase and calcium, Akt and retinol signaling, cytoskeletal elements, ATPases, ion channels, protein synthesis and the proteasome system, intermediary, nucleic acid and lipid metabolism, huntingtin, guidance cues, transforming growth factor beta-1 and hepatocyte nuclear factor 4-alpha. The network identified involves (a) cellular assembly and organization and (b) cell signaling and cell cycle, suggesting that Gls is crucial for neuronal maturation.
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- 2013
9. Developing Spindlin1 small-molecule inhibitors by using protein microarrays
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Jianjun Shen, Mahmoud I. Khalil, Jie Lv, Mark T. Bedford, Xue Bai, Gianluca Sbardella, Xiaonan Su, Monica Viviano, Narkhyun Bae, Sabrina Castellano, Haitao Li, Cari A. Sagum, Donghang Cheng, Kaifu Chen, and Claire Johnson
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0301 basic medicine ,Models, Molecular ,Microarray ,Druggability ,Protein Array Analysis ,Cell Cycle Proteins ,Biology ,Article ,Small Molecule Libraries ,03 medical and health sciences ,Methyllysine ,chemistry.chemical_compound ,Structure-Activity Relationship ,Piperidines ,Humans ,Epigenetics ,Molecular Biology ,X-ray crystallography ,030102 biochemistry & molecular biology ,Dose-Response Relationship, Drug ,Molecular Structure ,Effector ,Small molecules ,Cell Biology ,Phosphoproteins ,Small molecule ,Cell biology ,Post-translational modifications ,Screening ,030104 developmental biology ,chemistry ,Benzamides ,Protein microarray ,H3K4me3 ,Microtubule-Associated Proteins - Abstract
The discovery of inhibitors of methyl- and acetyl-binding domains has provided evidence for the 'druggability' of epigenetic effector molecules. The small-molecule probe UNC1215 prevents methyl-dependent protein-protein interactions by engaging the aromatic cage of MBT domains and, with lower affinity, Tudor domains. Using a library of tagged UNC1215 analogs, we screened a protein-domain microarray of human methyllysine effector molecules to rapidly detect compounds with new binding profiles with either increased or decreased specificity. Using this approach, we identified a compound (EML405) that acquired a novel interaction with the Tudor-domain-containing protein Spindlin1 (SPIN1). Structural studies facilitated the rational synthesis of SPIN1 inhibitors with increased selectivity (EML631-633), which engage SPIN1 in cells, block its ability to 'read' H3K4me3 marks and inhibit its transcriptional-coactivator activity. Protein microarrays can thus be used as a platform to 'target-hop' and identify small molecules that bind and compete with domain-motif interactions.
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- 2016
10. Peptide toxin glacontryphan-M is present in the wings of the butterfly Hebomoia glaucippe (Linnaeus, 1758) (Lepidoptera: Pieridae)
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Martin Lödl, Gert Lubec, Narkhyun Bae, and Lin Li
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animal structures ,Sequence analysis ,Blotting, Western ,Molecular Sequence Data ,Mollusk Venoms ,Peptide ,medicine.disease_cause ,Methylation ,Peptides, Cyclic ,Lepidoptera genitalia ,Tandem Mass Spectrometry ,Conus ,medicine ,Animals ,Wings, Animal ,Electrophoresis, Gel, Two-Dimensional ,Amino Acid Sequence ,Hebomoia glaucippe ,chemistry.chemical_classification ,Multidisciplinary ,Two-dimensional gel electrophoresis ,biology ,Toxin ,Biological Sciences ,biology.organism_classification ,Molecular biology ,chemistry ,Butterflies ,Protein Processing, Post-Translational ,Chromatography, Liquid ,Pieridae - Abstract
Protein profiling has revealed the presence of glacontryphan-M, a peptide toxin identified only in the sea snail genus Conus , in the wings of Hebomoia glaucippe (HG). The wings and body of HG were homogenized and the proteins were extracted and analyzed by 2D gel electrophoresis with subsequent in-gel digestion. Posttranslational protein modifications were detected and analyzed by nano–LC-MS/MS. An antibody was generated against glacontryphan-M, and protein extracts from the wings of HG samples from Malaysia, Indonesia, and the Philippines were tested by immunoblotting. Glacontryphan-M was unambiguously identified in the wings of HG containing the following posttranslational protein modifications: monoglutamylation at E55, methylation at E53, quinone modification at W61, cyanylation at C56, and amidation of the C terminus at G63. Immunoblotting revealed the presence of the toxin in the wings of HG from all origins, showing a single band for glacontryphan-M in HG samples from Malaysia and Philippines and a double band in HG samples from Indonesia. Intriguingly, sequence analysis indicated that the Conus glacontryphan is identical to that of HG. The toxin may function as a defense against diverse predators, including ants, mantes, spiders, lizards, green frogs, and birds.
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- 2012
11. Mass spectrometrical analysis of bilin-binding protein from the wing of Hebomoia glaucippe (Linnaeus, 1758) (Lepidoptera: Pieridae)
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Narkhyun Bae, Martin Lödl, Gert Lubec, and Arnold Pollak
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Hypusine ,Gel electrophoresis ,Apolipoprotein D ,biology ,Binding protein ,Clinical Biochemistry ,Lipocalin ,biology.organism_classification ,Biochemistry ,Analytical Chemistry ,chemistry.chemical_compound ,chemistry ,Hebomoia glaucippe ,Bilin ,Function (biology) - Abstract
Bilin-binding protein (BBP) is a member of the lipocalin superfamily and a pigment protein in Lepidoptera. It is binding to a series of lipidic compounds but its functions remain to be elucidated. Working on wing proteins in Hebomoia glaucippe, we observed this protein on gels and decided to characterize BBP. A gel-based mass spectrometrical method using two-dimensional gel electrophoresis followed by in-gel digestion of protein spots followed by nano-LC-ESI-MS/MS (ion trap, HCT) identification and characterization of proteins was applied. An antibody was generated against the protein and immunoblotting in the butterfly and mouse brain was carried out. Two spots were identified from the butterfly wing as BBP (P09464) with high sequence coverage. Nitrotyrosination (Y163; as aminotyrosine) was observed and nitration was verified using immunoblotting. Additional posttranslational modifications (PTMs) as hypusine, carboxylation, kynurenine, aminoadipic acid, were proposed. The presence of BBP-immunoreactive protein was also observed in mouse brain. The characterization of BBP showed high sequence similarity with mouse apolipoprotein D and the findings suggest a tentative function of BBP comparable to apolipoproteins. The role of the PTMs remains elusive but nitration, in analogy to nitration effects reported in literature, proposes a role for mechanoelastic proteins and protein-protein interactions.
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- 2012
12. Mass spectrometrical analysis of cuticular proteins from the wing of Hebemoia glaucippe (Linnaeus, 1758) (Lepidoptera: Pieridae)
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Narkhyun Bae, Gert Lubec, Arnold Pollak, and Martin Lödl
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Gene isoform ,animal structures ,media_common.quotation_subject ,Cuticle ,Molecular Sequence Data ,Biophysics ,Genes, Insect ,Peptide ,Insect ,Tandem mass spectrometry ,Biochemistry ,Tandem Mass Spectrometry ,Botany ,medicine ,Animals ,Protein Isoforms ,Wings, Animal ,Electrophoresis, Gel, Two-Dimensional ,Amino Acid Sequence ,media_common ,chemistry.chemical_classification ,Chymotrypsin ,biology ,fungi ,Trypsin ,biology.organism_classification ,chemistry ,biology.protein ,Insect Proteins ,Arthropod ,Butterflies ,Protein Processing, Post-Translational ,medicine.drug - Abstract
Although several insect cuticular genes and proteins are annotated and an arthropod cuticular database is available, mass spectrometrical data on cuticular proteins and their post-translational modifications are limited. Wings from Hebemoia glaucippe were analyzed by scanning electron microscopy or homogenized, proteins were extracted and run on 2DE. In-gel digestion was carried out by using trypsin, chymotrypsin and Asp-N and subsequently the resulting peptides and post-translational modifications were identified by ion trap tandem mass spectrometry (nano-LC-ESI-MS/MS; HCT). A complex wing skeleton and the cuticle of H. glaucippe were demonstrated. Cuticle protein 18.6, isoform A, pupal cuticle protein, cuticular protein CPR59A and two putative proteins, putative cuticular protein B2DBJ and putative cuticle protein CPG31 with two expression forms were identified. Two phosphorylation sites on the same peptide, T213 and S214, were identified on putative cuticle protein CPG31, quinone formation was observed at Y76 on cuticular protein CPR59A probably indicating the presence of post-translational modifications. The results may be relevant for the interpretation of mechanoelastic and physical properties of these proteins. Along with the extraordinary architecture the proteinaceous matrix is probably representing or allowing the unusual aerodynamic function of the butterfly wing. Moreover, the results may be important for mechanisms of insecticide and drought resistance.
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- 2011
13. An electrophoretic approach to screen for glutamine deamidation
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Jae-Won Yang, Harald H. Sitte, Javier Márquez, Arnold Pollak, Gert Lubec, and Narkhyun Bae
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Cell Extracts ,Tissue transglutaminase ,Glutamine ,Biophysics ,Buffers ,Cleavage (embryo) ,Biochemistry ,law.invention ,law ,Animals ,Humans ,Electrophoresis, Gel, Two-Dimensional ,Deamidation ,Molecular Biology ,Polyacrylamide gel electrophoresis ,Gel electrophoresis ,Membranes ,Chromatography ,Staining and Labeling ,biology ,Human Growth Hormone ,Chemistry ,Cell Biology ,Amides ,Rats ,Electrophoresis ,Receptor, Serotonin, 5-HT1A ,biology.protein ,Recombinant DNA ,Subcellular Fractions - Abstract
Protein deamidation is a posttranslational modification with important implications in physiology and medicine. There is, however, no simple technique for a rapid screening of protein deamidation. The deamidating activity of transglutaminase was applied to establish a simple method for the screen of protein deamidation using recombinant human growth hormone, a rat hippocampal membrane fraction, and a cell homogenate enriched in 5-hydroxytryptamine-1A receptor as model systems. Here we report a simple, economic, and fast approach to assess protein deamidation by two electrophoretic methods: differential cleavage on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) via in situ V8 protease digestion and the principle of spot shifting via blue native (BN)-PAGE/two-dimensional (2D)-SDS-PAGE/immunoblotting.
- Published
- 2012
14. A transcriptional coregulator, SPIN·DOC, attenuates the coactivator activity of Spindlin1.
- Author
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Narkhyun Bae, Min Gao, Xu Li, Premkumar, Tolkappiyan, Sbardella, Gianluca, Junjie Chen, and Bedford, Mark T.
- Subjects
- *
GENE regulatory networks , *EMBRYOLOGY , *GENETIC transcription , *HISTONE methylation , *CYTOKINE genetics - Abstract
Spindlin1 (SPIN1) is a transcriptional coactivator with critical functions in embryonic development and emerging roles in cancer. SPIN1 harbors three Tudor domains, two of which engage the tail of histone H3 by reading the H3-Lys-4 trimethylation and H3-Arg-8 asymmetric dimethylation marks. To gain mechanistic insight into how SPIN1 functions as a transcriptional coactivator, here we purified its interacting proteins. We identified an uncharacterized protein (C11orf84), which we renamed SPIN1 docking protein (SPIN·DOC), that directly binds SPIN1 and strongly disrupts its histone methylation reading ability, causing it to disassociate from chromatin. The Spindlin family of coactivators has five related members (SPIN1, 2A, 2B, 3, and 4), and we found that all of them bind SPIN·DOC. It has been reported previously that SPIN1 regulates gene expression in the Wnt signaling pathway by directly interacting with transcription factor 4 (TCF4). We observed here that SPIN·DOC associates with TCF4 in a SPIN1-dependent manner and dampens SPIN1 coactivator activity in TOPflash reporter assays. Furthermore, knockdown and overexpression experiments indicated that SPIN.DOC represses the expression of a number of SPIN1- regulated genes, including those encoding ribosomalRNAand the cytokine IL1B. In conclusion, we have identified SPIN·DOC as a transcriptional repressor that binds SPIN1 and masks its ability to engage the H3-Lys-4 trimethylation activation mark. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
15. Strain-dependent expression of metabolic proteins in the mouse hippocampus.
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Pollak, Daniela D., Narkhyun Bae, Mostafa, Gehan, and Hoeger, Harald
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HIPPOCAMPUS (Brain) , *LABORATORY mice , *COGNITIVE ability , *PROTEINS , *GEL electrophoresis , *MASS spectrometry - Abstract
Individual mouse strains differ significantly in terms of behavior and cognitive function. Strain-specific variation of metabolic protein levels in the hippocampus among various commonly used mouse strains, however, has not been investigated yet. A proteomic approach based on two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry [high capacity ion trap (HCT)] has been chosen to address this question by determining strain-dependent levels of metabolic proteins in hippocampal tissue of four inbred and one outbred mouse strain. Statistical analysis of protein spots on 2-DE gels of the individual strains ( n = 10) revealed significant strain-dependent differences in densities of 39 spots. Subsequent HCT analysis led to the identification of 22 different metabolic proteins presenting with differential protein levels among the five mouse strains investigated. Among those are proteins concerned with the metabolism of amino acid, nucleic acid, carbohydrate and energy. Moreover, proteins known to play a pivotal role in the processes of learning and memory, such as calcium/calmodulin- dependent protein kinase type II alpha chain, were found to present with significant inter-strain variability, which is also in agreement with our previous reports. Strain-specific protein levels of metabolic proteins in the mouse hippocampus may provide some insight into the molecular underpinnings and genetic determination of strain-dependent neuronal function. Furthermore, data presented herein emphasize the significance of the genetic background for the analysis of metabolic pathways in the hippocampus in wild-type mice as well as in gene-targeting experiments. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
16. MOESM4 of Histone peptide microarray screen of chromo and Tudor domains defines new histone lysine methylation interactions
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Shanle, Erin, Shinsky, Stephen, Bridgers, Joseph, Narkhyun Bae, Sagum, Cari, Krajewski, Krzysztof, Rothbart, Scott, Bedford, Mark, and Strahl, Brian
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3. Good health - Abstract
Additional file 4: Figure S2. Peptide microarray data for SGF29 and Taf3. Representative array images of A) SGF29 double Tudor domain and Taf3 PHD domain showing peptide binding indicated in red (right panel). The peptide tracer is shown in green (left panel). Positive antibody controls are outlined in white.
17. MOESM8 of Histone peptide microarray screen of chromo and Tudor domains defines new histone lysine methylation interactions
- Author
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Shanle, Erin, Shinsky, Stephen, Bridgers, Joseph, Narkhyun Bae, Sagum, Cari, Krajewski, Krzysztof, Rothbart, Scott, Bedford, Mark, and Strahl, Brian
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3. Good health - Abstract
Additional file 8: Figure S6. TDRD3 Tudor domain histone peptide microarray. A) Representative array images of TDRD3 Tudor domain showing peptide binding indicated in red (right panel). The peptide tracer is shown in green (left panel).
18. Histone peptide microarray screen of chromo and Tudor domains defines new histone lysine methylation interactions
- Author
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Cari A. Sagum, Mark T. Bedford, Erin K. Shanle, Joseph B. Bridgers, Krzysztof Krajewski, Scott B. Rothbart, Stephen A. Shinsky, Narkhyun Bae, and Brian D. Strahl
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0301 basic medicine ,Tudor domain ,Chromosomal Proteins, Non-Histone ,Histone lysine methylation ,Protein Array Analysis ,Cell Cycle Proteins ,Computational biology ,Biology ,Methylation ,Histone methylation ,Histones ,03 medical and health sciences ,Histone H1 ,Histone H2A ,Genetics ,Humans ,Histone code ,Protein Interaction Domains and Motifs ,Molecular Biology ,Peptide microarray ,Tudor Domain ,Research ,Lysine ,Phosphoproteins ,Chromatin ,Chromodomain ,HEK293 Cells ,030104 developmental biology ,Histone ,Chromobox Protein Homolog 5 ,Histone methyltransferase ,biology.protein ,Heterochromatin protein 1 ,Microtubule-Associated Proteins - Abstract
Background Histone posttranslational modifications (PTMs) function to regulate chromatin structure and function in part through the recruitment of effector proteins that harbor specialized “reader” domains. Despite efforts to elucidate reader domain–PTM interactions, the influence of neighboring PTMs and the target specificity of many reader domains is still unclear. The aim of this study was to use a high-throughput histone peptide microarray platform to interrogate 83 known and putative histone reader domains from the chromo and Tudor domain families to identify their interactions and characterize the influence of neighboring PTMs on these interactions. Results Nearly a quarter of the chromo and Tudor domains screened showed interactions with histone PTMs by peptide microarray, revealing known and several novel methyllysine interactions. Specifically, we found that the CBX/HP1 chromodomains that recognize H3K9me also recognize H3K23me2/3—a poorly understood histone PTM. We also observed that, in addition to their interaction with H3K4me3, Tudor domains of the Spindlin family also recognized H4K20me3—a previously uncharacterized interaction. Several Tudor domains also showed novel interactions with H3K4me as well. Conclusions These results provide an important resource for the epigenetics and chromatin community on the interactions of many human chromo and Tudor domains. They also provide the basis for additional studies into the functional significance of the novel interactions that were discovered. Electronic supplementary material The online version of this article (doi:10.1186/s13072-017-0117-5) contains supplementary material, which is available to authorized users.
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19. MOESM8 of Histone peptide microarray screen of chromo and Tudor domains defines new histone lysine methylation interactions
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Shanle, Erin, Shinsky, Stephen, Bridgers, Joseph, Narkhyun Bae, Sagum, Cari, Krajewski, Krzysztof, Rothbart, Scott, Bedford, Mark, and Strahl, Brian
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3. Good health - Abstract
Additional file 8: Figure S6. TDRD3 Tudor domain histone peptide microarray. A) Representative array images of TDRD3 Tudor domain showing peptide binding indicated in red (right panel). The peptide tracer is shown in green (left panel).
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