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  1. Article: Proteomic Interactome of C. elegans Mediator Complex Subunit 28 (MDT-28) Reveals Predominant Association with a Restricted Set of Core Mediator Subunits and an Affinity to Additional Structural and Enzymatic Proteins.

    Yilma, P / Kostrouchová, M / Talacko, P / Kostrouchová, V / Kostrouch, D / Novák, P

    Folia biologica

    2020  Volume 65, Issue 5-6, Page(s) 203–211

    Abstract: Transcription factors exert their regulatory potential on RNA polymerase II machinery through a multiprotein complex called Mediator complex or Mediator. The Mediator complex integrates regulatory signals from cell regulatory cascades with the regulation ...

    Abstract Transcription factors exert their regulatory potential on RNA polymerase II machinery through a multiprotein complex called Mediator complex or Mediator. The Mediator complex integrates regulatory signals from cell regulatory cascades with the regulation by transcription factors. The Mediator complex consists of 25 subunits in Saccharomyces cerevisiae and 30 or more subunits in multicellular eukaryotes. Mediator subunit 28 (MED28), along with MED30, MED23, MED25 and MED26, belong to presumably evolutionarily new subunits that seem to be absent in unicellular eukaryotes and are likely to have evolved together with multicellularity and cell differentiation. Previously, we have shown that an originally uncharacterized predicted gene, F28F8.5, is the true MED28 orthologue in Caenorhabditis elegans (mdt-28) and showed that it is involved in a spectrum of developmental processes. Here, we studied the proteomic interactome of MDT-28 edited as GFP::MDT-28 using Crispr/Cas9 technology or MDT-28::GFP expressed from extrachromosomal arrays in transgenic C. elegans exploiting the GFPTRAP system and mass spectrometry. The results show that MDT-28 associates with the Head module subunits MDT-6, MDT-8, MDT-11, MDT-17, MDT- 20, MDT-22, and MDT-30 and the Middle module subunit MDT-14. The analyses also identified additional proteins as preferential MDT-28 interactants, including chromatin-organizing proteins, structural proteins and enzymes. The results provide evidence for MDT-28 engagement in the Mediator Head module and support the possibility of physical (direct or indirect) interaction of MDT-28 with additional proteins, reflecting the transcription-regulating potential of primarily structural and enzymatic proteins at the level of the Mediator complex.
    MeSH term(s) Alleles ; Animals ; Caenorhabditis elegans/metabolism ; Caenorhabditis elegans Proteins/metabolism ; Mediator Complex/metabolism ; Nuclear Proteins/metabolism ; Protein Binding ; Protein Subunits/metabolism ; Proteomics
    Chemical Substances Caenorhabditis elegans Proteins ; MDT-28 protein, C elegans ; Mediator Complex ; Nuclear Proteins ; Protein Subunits
    Language English
    Publishing date 2020-04-07
    Publishing country Czech Republic
    Document type Journal Article
    ZDB-ID 419223-0
    ISSN 0015-5500
    ISSN 0015-5500
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

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  2. Article: Valproic Acid Decreases the Nuclear Localization of MDT-28, the Nematode Orthologue of MED28.

    Kostrouchová, M / Kostrouchová, V / Yilma, P / Benda, A / Mandys, V

    Folia biologica

    2018  Volume 64, Issue 1, Page(s) 1–9

    Abstract: Mediator is a multiprotein complex that connects regulation mediated by transcription factors with RNA polymerase II transcriptional machinery and integrates signals from the cell regulatory cascades with gene expression. One of the Mediator subunits, ... ...

    Abstract Mediator is a multiprotein complex that connects regulation mediated by transcription factors with RNA polymerase II transcriptional machinery and integrates signals from the cell regulatory cascades with gene expression. One of the Mediator subunits, Mediator complex subunit 28 (MED28), has a dual nuclear and cytoplasmic localization and function. In the nucleus, MED28 functions as part of Mediator and in the cytoplasm, it interacts with cytoskeletal proteins and is part of the regulatory cascades including that of Grb2. MED28 thus has the potential to bring cytoplasmic regulatory interactions towards the centre of gene expression regulation. In this study, we identified MDT-28, the nematode orthologue of MED28, as a likely target of lysine acetylation using bioinformatic prediction of posttranslational modifications. Lysine acetylation was experimentally confirmed using anti-acetyl lysine antibody on immunoprecipitated GFP::MDT-28 expressed in synchronized C. elegans. Valproic acid (VPA), a known inhibitor of lysine deacetylases, enhanced the lysine acetylation of GFP::MDT-28. At the subcellular level, VPA decreased the nuclear localization of GFP::MDT-28 detected by fluorescencelifetime imaging microscopy (FLIM). This indicates that the nuclear pool of MDT-28 is regulated by a mechanism sensitive to VPA and provides an indirect support for a variable relative proportion of MED28 orthologues with other Mediator subunits.
    MeSH term(s) Acetylation ; Amino Acid Sequence ; Animals ; Caenorhabditis elegans/drug effects ; Caenorhabditis elegans/metabolism ; Caenorhabditis elegans Proteins/chemistry ; Caenorhabditis elegans Proteins/metabolism ; Cell Nucleus/drug effects ; Cell Nucleus/metabolism ; Computational Biology ; Densitometry ; Green Fluorescent Proteins/metabolism ; Humans ; Larva/drug effects ; Lysine/metabolism ; Mediator Complex/chemistry ; Mediator Complex/metabolism ; Nuclear Proteins/chemistry ; Nuclear Proteins/metabolism ; Protein Transport/drug effects ; Recombinant Fusion Proteins/metabolism ; Sequence Homology, Amino Acid ; Valproic Acid/pharmacology
    Chemical Substances Caenorhabditis elegans Proteins ; MDT-28 protein, C elegans ; Mediator Complex ; Nuclear Proteins ; Recombinant Fusion Proteins ; Green Fluorescent Proteins (147336-22-9) ; Valproic Acid (614OI1Z5WI) ; Lysine (K3Z4F929H6)
    Language English
    Publishing date 2018-03-30
    Publishing country Czech Republic
    Document type Journal Article
    ZDB-ID 419223-0
    ISSN 0015-5500
    ISSN 0015-5500
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    In stock of ZB MED Cologne/Königswinter

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  3. Article: SMED-TLX-1 (NR2E1) is critical for tissue and body plan maintenance in Schmidtea mediterranea in fasting/feeding cycles.

    Raška, O / Kostrouchová, V / Behenský, F / Yilma, P / Saudek, V / Kostrouch, Z / Kostrouchová, M

    Folia biologica

    2011  Volume 57, Issue 6, Page(s) 223–231

    Abstract: Nuclear receptors (NRs), or nuclear hormone receptors (NHRs), are transcription factors that regulate development and metabolism of most if not all animal species. Their regulatory networks include ... ...

    Abstract Nuclear receptors (NRs), or nuclear hormone receptors (NHRs), are transcription factors that regulate development and metabolism of most if not all animal species. Their regulatory networks include conserved mechanisms that are shared in-between species as well as mechanisms that are restricted to certain phyla or even species. In search for conserved members of the NHR family in Schmidtea mediterranea, we identified a molecular signature of a class of NRs, NR2E1, in the S. mediterranea genome and cloned its complete cDNA coding sequence. The derived amino acid sequence shows a high degree of conservation of both DNA-binding domain and ligand- binding domain and a remarkably high homology to vertebrate NR2E1 and C. elegans NHR-67. Quantitative PCR detected approximately ten-fold higher expression of Smed-tlx-1 in the proximal part of the head compared to the tail region. The expression of Smed-tlx-1 is higher during fed state than during fasting. Smed-tlx-1 down-regulation by RNA interference affects the ability of the animals to maintain body plan and induces defects of brain, eyes and body shape during fasting and re-growing cycles. These results suggest that SMED-TLX-1 is critical for tissue and body plan maintenance in planaria.
    MeSH term(s) Amino Acid Sequence ; Animals ; Body Patterning/genetics ; Cloning, Molecular ; Fasting/physiology ; Feeding Behavior/physiology ; Gene Expression Regulation ; Helminth Proteins/chemistry ; Helminth Proteins/genetics ; Helminth Proteins/metabolism ; Humans ; Molecular Sequence Data ; Organ Specificity/genetics ; Phylogeny ; RNA Interference ; Receptors, Cytoplasmic and Nuclear/chemistry ; Receptors, Cytoplasmic and Nuclear/genetics ; Receptors, Cytoplasmic and Nuclear/metabolism ; Sequence Alignment ; Turbellaria/embryology ; Turbellaria/genetics ; Turbellaria/physiology
    Chemical Substances Helminth Proteins ; Receptors, Cytoplasmic and Nuclear
    Language English
    Publishing date 2011-05-05
    Publishing country Czech Republic
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 419223-0
    ISSN 0015-5500
    ISSN 0015-5500
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  4. Article ; Online: SKIP and BIR-1/Survivin have potential to integrate proteome status with gene expression.

    Kostrouch, David / Kostrouchová, Markéta / Yilma, Petr / Chughtai, Ahmed Ali / Novotný, Jan Philipp / Novák, Petr / Kostrouchová, Veronika / Kostrouchová, Marta / Kostrouch, Zdeněk

    Journal of proteomics

    2014  Volume 110, Page(s) 93–106

    Abstract: SKIP and BIR are evolutionarily conserved proteins; SKIP (SKP-1) is a known transcription and splicing cofactor while BIR-1/Survivin regulates cell division, gene expression and development. Their loss of function induces overlapping developmental ... ...

    Abstract SKIP and BIR are evolutionarily conserved proteins; SKIP (SKP-1) is a known transcription and splicing cofactor while BIR-1/Survivin regulates cell division, gene expression and development. Their loss of function induces overlapping developmental phenotypes. We searched for SKP-1 and BIR-1 interaction on protein level using yeast two-hybrid screens and identified partially overlapping categories of proteins as SKIP-1 and BIR-1 interactors. The interacting proteins included ribosomal proteins, transcription factors, translation factors and cytoskeletal and motor proteins suggesting involvement in multiple protein complexes. To visualize the effect of BIR-1 on the proteome in Caenorhabditis elegans we induced a short time pulse BIR-1 overexpression in synchronized L1 larvae. This led to a dramatic alteration of the whole proteome pattern indicating that BIR-1 alone has the capacity to alter the chromatographic profile of many target proteins including proteins found to be interactors in yeast two hybrid screens. The results were validated for ribosomal proteins RPS3 and RPL5, non-muscle myosin and TAC-1, a transcription cofactor and a centrosome associated protein. Together, these results suggest that SKP-1 and BIR-1 are multifunctional proteins that form multiple protein complexes in both shared and distinct pathways and have the potential to connect proteome signals with the regulation of gene expression.
    Biological significance: The genomic organization of the genes encoding BIR-1 and SKIP (SKP-1) in C. elegans have suggested that these two factors, each evolutionarily conserved, have related functions. However, these functional connections have remained elusive and underappreciated in light of limited information from C. elegans and other biological systems. Our results provide further evidence for a functional link between these two factors and suggest they may transmit proteome signals towards the regulation of gene expression.
    MeSH term(s) Animals ; Caenorhabditis elegans/metabolism ; Caenorhabditis elegans Proteins/metabolism ; Gene Expression Regulation/physiology ; Nuclear Proteins/metabolism ; Proteome/metabolism ; Signal Transduction/physiology ; Survivors ; Ubiquitin-Protein Ligase Complexes
    Chemical Substances Caenorhabditis elegans Proteins ; Nuclear Proteins ; Proteome ; bir-1 protein, C elegans ; Ubiquitin-Protein Ligase Complexes (EC 2.3.2.23) ; skp-1 protein, C elegans (EC 2.3.2.23)
    Language English
    Publishing date 2014-08-01
    Publishing country Netherlands
    Document type Journal Article ; Research Support, N.I.H., Intramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2400835-7
    ISSN 1876-7737 ; 1874-3919
    ISSN (online) 1876-7737
    ISSN 1874-3919
    DOI 10.1016/j.jprot.2014.07.023
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  5. Article ; Online: GEI-8, a homologue of vertebrate nuclear receptor corepressor NCoR/SMRT, regulates gonad development and neuronal functions in Caenorhabditis elegans.

    Mikoláš, Pavol / Kollárová, Johana / Sebková, Kateřina / Saudek, Vladimír / Yilma, Petr / Kostrouchová, Markéta / Krause, Michael W / Kostrouch, Zdenek / Kostrouchová, Marta

    PloS one

    2013  Volume 8, Issue 3, Page(s) e58462

    Abstract: NCoR and SMRT are two paralogous vertebrate proteins that function as corepressors with unliganded nuclear receptors. Although C. elegans has a large number of nuclear receptors, orthologues of the corepressors NCoR and SMRT have not unambiguously been ... ...

    Abstract NCoR and SMRT are two paralogous vertebrate proteins that function as corepressors with unliganded nuclear receptors. Although C. elegans has a large number of nuclear receptors, orthologues of the corepressors NCoR and SMRT have not unambiguously been identified in Drosophila or C. elegans. Here, we identify GEI-8 as the closest homologue of NCoR and SMRT in C. elegans and demonstrate that GEI-8 is expressed as at least two isoforms throughout development in multiple tissues, including neurons, muscle and intestinal cells. We demonstrate that a homozygous deletion within the gei-8 coding region, which is predicted to encode a truncated protein lacking the predicted NR domain, results in severe mutant phenotypes with developmental defects, slow movement and growth, arrested gonadogenesis and defects in cholinergic neurotransmission. Whole genome expression analysis by microarrays identified sets of de-regulated genes consistent with both the observed mutant phenotypes and a role of GEI-8 in regulating transcription. Interestingly, the upregulated transcripts included a predicted mitochondrial sulfide:quinine reductase encoded by Y9C9A.16. This locus also contains non-coding, 21-U RNAs of the piRNA class. Inhibition of the expression of the region coding for 21-U RNAs leads to irregular gonadogenesis in the homozygous gei-8 mutants, but not in an otherwise wild-type background, suggesting that GEI-8 may function in concert with the 21-U RNAs to regulate gonadogenesis. Our results confirm that GEI-8 is the orthologue of the vertebrate NCoR/SMRT corepressors and demonstrate important roles for this putative transcriptional corepressor in development and neuronal function.
    MeSH term(s) Amino Acid Sequence ; Animals ; Base Sequence ; Caenorhabditis elegans/genetics ; Caenorhabditis elegans/physiology ; Caenorhabditis elegans Proteins/genetics ; Caenorhabditis elegans Proteins/metabolism ; Co-Repressor Proteins/genetics ; Co-Repressor Proteins/metabolism ; Gene Deletion ; Gene Expression Profiling ; Gene Expression Regulation/genetics ; Gonads/growth & development ; Microarray Analysis ; Molecular Sequence Data ; Neurons/physiology ; Nuclear Receptor Co-Repressor 1/genetics ; Nuclear Receptor Co-Repressor 2/genetics ; Receptors, Cytoplasmic and Nuclear/metabolism ; Sequence Alignment ; Sequence Analysis, DNA
    Chemical Substances Caenorhabditis elegans Proteins ; Co-Repressor Proteins ; GEI-8 protein, C elegans ; Nuclear Receptor Co-Repressor 1 ; Nuclear Receptor Co-Repressor 2 ; Receptors, Cytoplasmic and Nuclear
    Language English
    Publishing date 2013-03-06
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1932-6203
    ISSN (online) 1932-6203
    DOI 10.1371/journal.pone.0058462
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  6. Article: Elevated and deregulated expression of HDAC3 in human astrocytic glial tumours.

    Libý, P / Kostrouchová, M / Pohludka, M / Yilma, P / Hrabal, P / Sikora, J / Brozová, E / Rall, J E / Kostrouch, Z

    Folia biologica

    2006  Volume 52, Issue 1-2, Page(s) 21–33

    Abstract: Abnormal expression of histone deacetylases may contribute to the establishment of a cancer specific transcription profile. We examined expression of HDAC3 in human non-malignant gliosis and glial astrocytic tumours. Samples from four non-malignant ... ...

    Abstract Abnormal expression of histone deacetylases may contribute to the establishment of a cancer specific transcription profile. We examined expression of HDAC3 in human non-malignant gliosis and glial astrocytic tumours. Samples from four non-malignant gliosis and 17 astrocytic gliomas (six of grade II, one of grade III and ten of grade IV) removed for therapeutic purposes were assayed for HDAC3 expression at mRNA and protein levels. HDAC3 mRNA was detected in non-tumorous gliosis as well as in all examined glial tumours. Seven out of eleven examined high-grade tumours showed an elevated number of copies of HDAC3 mRNA. Western blot analysis detected high levels of expression of HDAC3 in the majority of the examined tumours. Immunohistochemistry and immunofluorescence made on a collection of 35 astrocytic tumours detected nuclear as well as cytoplasmic HDAC3 expression in all of those tumours. While the distribution of HDAC3 was both nuclear as well as cytoplasmic and moderate in intensity in non-malignant tissues and low-grade gliomas, high-grade tumours expressed HDAC3 in a focally deregulated pattern that included strongly pronounced cytoplasmic localization. Confocal microscopy and additional co-localization analysis detected nuclear HDAC3 in all tumours examined. We conclude that HDAC3 expression is elevated in human astrocytic tumours and its expression pattern is deregulated at the cellular level in high-grade gliomas.
    MeSH term(s) Amino Acid Sequence ; Astrocytoma/enzymology ; Astrocytoma/genetics ; Brain Neoplasms/enzymology ; Brain Neoplasms/genetics ; Cell Nucleus/metabolism ; Gene Expression Regulation, Neoplastic ; Gliosis/metabolism ; Histone Deacetylases/genetics ; Histone Deacetylases/metabolism ; Humans ; Microscopy, Confocal ; Molecular Sequence Data ; Protein Isoforms/genetics ; Protein Isoforms/metabolism ; RNA, Messenger/metabolism
    Chemical Substances Protein Isoforms ; RNA, Messenger ; Histone Deacetylases (EC 3.5.1.98) ; histone deacetylase 3 (EC 3.5.1.98)
    Language English
    Publishing date 2006
    Publishing country Czech Republic
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 419223-0
    ISSN 0015-5500
    ISSN 0015-5500
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  7. Article ; Online: Proteomic analysis uncovers a metabolic phenotype in C. elegans after nhr-40 reduction of function.

    Pohludka, Michal / Simeckova, Katerina / Vohanka, Jaroslav / Yilma, Petr / Novak, Petr / Krause, Michael W / Kostrouchova, Marta / Kostrouch, Zdenek

    Biochemical and biophysical research communications

    2008  Volume 374, Issue 1, Page(s) 49–54

    Abstract: Caenorhabditis elegans has an unexpectedly large number (284) of genes encoding nuclear hormone receptors, most of which are nematode-specific and are of unknown function. We have exploited comparative two-dimensional chromatography of synchronized ... ...

    Abstract Caenorhabditis elegans has an unexpectedly large number (284) of genes encoding nuclear hormone receptors, most of which are nematode-specific and are of unknown function. We have exploited comparative two-dimensional chromatography of synchronized cultures of wild type C. elegans larvae and a mutant in nhr-40 to determine if proteomic approaches will provide additional insight into gene function. Chromatofocusing, followed by reversed-phase chromatography and mass spectrometry, identified altered chromatographic patterns for a set of proteins, many of which function in muscle and metabolism. Prompted by the proteomic analysis, we find that the penetrance of the developmental phenotypes in the mutant is enhanced at low temperatures and by food restriction. The combination of our phenotypic and proteomic analysis strongly suggests that NHR-40 provides a link between metabolism and muscle development. Our results highlight the utility of comparative two-dimensional chromatography to provide a relatively rapid method to gain insight into gene function.
    MeSH term(s) Animals ; Caenorhabditis elegans/genetics ; Caenorhabditis elegans/metabolism ; Caenorhabditis elegans Proteins/analysis ; Caenorhabditis elegans Proteins/metabolism ; Chromatography, Liquid/methods ; Muscle Development/genetics ; Proteome/analysis ; Proteome/metabolism ; Proteomics/methods ; Receptors, Cytoplasmic and Nuclear/genetics ; Receptors, Cytoplasmic and Nuclear/physiology
    Chemical Substances Caenorhabditis elegans Proteins ; Nhr-40 protein, C elegans ; Proteome ; Receptors, Cytoplasmic and Nuclear
    Language English
    Publishing date 2008-07-09
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, N.I.H., Intramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 205723-2
    ISSN 1090-2104 ; 0006-291X ; 0006-291X
    ISSN (online) 1090-2104 ; 0006-291X
    ISSN 0006-291X
    DOI 10.1016/j.bbrc.2008.06.115
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