The Experts below are selected from a list of 798 Experts worldwide ranked by ideXlab platform

Dominique Mornet - One of the best experts on this subject based on the ideXlab platform.

  • Nuclear and nuclear envelope localization of dystrophin Dp71 and dystrophin-associated proteins (DAPs) in the C2C12 muscle cells: DAPs nuclear localization is modulated during myogenesis.
    Journal of cellular biochemistry, 2008
    Co-Authors: Ricardo González-ramírez, Dominique Mornet, Sara Luz Morales-lázaro, Victor Tapia-ramírez, Bulmaro Cisneros
    Abstract:

    Dystrophin and dystrophin-associated proteins (DAPs) form a complex around the sarcolemma, which gives stability to the sarcolemma and leads signal transduction. Recently, the nuclear presence of dystrophin Dp71 and DAPs has been revealed in different non-muscle cell types, opening the possibility that these proteins could also be present in the nucleus of muscle cells. In this study, we analyzed by Immunofluorescence assays and Immunoblotting analysis of cell fractions the subcellular localization of Dp71 and DAPs in the C(2)C(12) muscle cell line. We demonstrated the presence of Dp71, alpha-sarcoglycan, alpha-dystrobrevin, Beta-Dystroglycan and alpha-syntrophin not only in plasma membrane but also in the nucleus of muscle cells. In addition, we found by Immunoprecipitation assays that these proteins form a nuclear complex. Interestingly, myogenesis modulates the presence and/or relative abundance of DAPs in the plasma membrane and nucleus as well as the composition of the nuclear complex. Finally, we demonstrated the presence of Dp71, alpha-sarcoglycan, Beta-Dystroglycan, alpha-dystrobrevin and alpha-syntrophin in the C(2)C(12) nuclear envelope fraction. Interestingly, alpha-sarcoglycan and Beta-Dystroglycan proteins showed enrichment in the nuclear envelope, compared with the nuclear fraction, suggesting that they could function as inner nuclear membrane proteins underlying the secondary association of Dp71 and the remaining DAPs to the nuclear envelope. Nuclear envelope localization of Dp71 and DAPs might be involved in the nuclear envelope-associated functions, such as nuclear structure and modulation of nuclear processes. J. Cell. Biochem. 105: 735-745, 2008. (c) 2008 Wiley-Liss, Inc.

  • Nuclear and nuclear envelope localization of dystrophin Dp71 and dystrophin-associated proteins (DAPs) in the C(2)C(12) muscle cells: DAPs nuclear localization is modulated during myogenesis.
    Journal of Cellular Biochemistry, 2008
    Co-Authors: Ricardo González-ramírez, Dominique Mornet, Sara Luz Morales-lázaro, Victor Tapia-ramírez, Bulmaro Cisneros
    Abstract:

    Dystrophin and dystrophin-associated proteins (DAPs) form a complex around the sarcolemma, which gives stability to the sarcolemma and leads signal transduction. Recently, the nuclear presence of dystrophin Dp71 and DAPs has been revealed in different non-muscle cell types, opening the possibility that these proteins could also be present in the nucleus of muscle cells. In this study, we analyzed by Immunofluorescence assays and Immunoblotting analysis of cell fractions the subcellular localization of Dp71 and DAPs in the C(2)C(12) muscle cell line. We demonstrated the presence of Dp71, alpha-sarcoglycan, alpha-dystrobrevin, Beta-Dystroglycan and alpha-syntrophin not only in plasma membrane but also in the nucleus of muscle cells. In addition, we found by Immunoprecipitation assays that these proteins form a nuclear complex. Interestingly, myogenesis modulates the presence and/or relative abundance of DAPs in the plasma membrane and nucleus as well as the composition of the nuclear complex. Finally, we demonstrated the presence of Dp71, alpha-sarcoglycan, Beta-Dystroglycan, alpha-dystrobrevin and alpha-syntrophin in the C(2)C(12) nuclear envelope fraction. Interestingly, alpha-sarcoglycan and Beta-Dystroglycan proteins showed enrichment in the nuclear envelope, compared with the nuclear fraction, suggesting that they could function as inner nuclear membrane proteins underlying the secondary association of Dp71 and the remaining DAPs to the nuclear envelope. Nuclear envelope localization of Dp71 and DAPs might be involved in the nuclear envelope-associated functions, such as nuclear structure and modulation of nuclear processes. J. Cell. Biochem. 105: 735-745, 2008. (c) 2008 Wiley-Liss, Inc.

  • dp71 utrophin and Beta Dystroglycan expression and distribution in pc12 l6 cell cocultures
    Neuroreport, 2007
    Co-Authors: Ramses Ilarrazalomeli, Dominique Mornet, Bulmaro Cisnerosvega, Maria De Lourdes Cervantesgomez, Cecilia Montanez
    Abstract:

    Function of dystrophin Dp71 isoforms is unknown but seems related to neurite outgrowth and synapse formation. To evaluate Dp71 role in myoneural synapses, we established a coculture model using PC12 cells and L6 myotubes and analyzed expression and localization of Dp71 and related proteins, utrophin and Beta-Dystroglycan, in PC12 cells. Confocal microscopy showed Dp71d isoform in PC12 nuclei, golgi-complex-like and endoplasmic reticulum-like structures, whereas Dp71ab concentrates at neurite tips and cytoplasm, colocalizing with Beta-Dystroglycan, utrophin, synaptophysin and acetylcholine receptors. Evidences suggest that Dp71ab isoform, unlike Dp71d, may take part in neurite-related processes. This is the first work on Dp and members of Dp-associated protein complex roles in a cell-line based coculturing system, which may be useful in determining Dp71 isoforms associations.

  • Dp71, utrophin and Beta-Dystroglycan expression and distribution in PC12/L6 cell cocultures.
    NeuroReport, 2007
    Co-Authors: Ramses Ilarraza-lomeli, Dominique Mornet, Bulmaro Cisneros-vega, Maria De Lourdes Cervantes-gomez, Cecilia Montanez
    Abstract:

    Function of dystrophin Dp71 isoforms is unknown but seems related to neurite outgrowth and synapse formation. To evaluate Dp71 role in myoneural synapses, we established a coculture model using PC12 cells and L6 myotubes and analyzed expression and localization of Dp71 and related proteins, utrophin and Beta-Dystroglycan, in PC12 cells. Confocal microscopy showed Dp71d isoform in PC12 nuclei, golgi-complex-like and endoplasmic reticulum-like structures, whereas Dp71ab concentrates at neurite tips and cytoplasm, colocalizing with Beta-Dystroglycan, utrophin, synaptophysin and acetylcholine receptors. Evidences suggest that Dp71ab isoform, unlike Dp71d, may take part in neurite-related processes. This is the first work on Dp and members of Dp-associated protein complex roles in a cell-line based coculturing system, which may be useful in determining Dp71 isoforms associations.

  • Dp71ab/DAPs complex composition changes during the differentiation process in PC12 cells.
    Journal of Cellular Biochemistry, 2007
    Co-Authors: Jose Romo-yanez, Victor Ceja, Ramses Ilarraza-lomeli, Ramon Coral-vazquez, Francisco Velazquez, Dominique Mornet, Alvaro Rendon, Cecilia Montanez
    Abstract:

    PC12 cells express different Dp71 isoforms originated from alternative splicing; one of them, Dp71ab lacks exons 71 and 78. To gain insight into the function of Dp71 isoforms we identified dystrophin associated proteins (DAPs) that associate in vivo with Dp71ab during nerve growth factor (NGF) induced differentiation of PC12 cells. DAPs expression was analyzed by RT-PCR, Western blot and indirect immunofluorescence, showing the presence of each mRNA and protein corresponding to alpha-, Beta-, gamma-, delta-, and epsilon-sarcoglycans as well as zeta-sarcoglycan mRNA. Western blot analysis also revealed the expression of Beta-Dystroglycan, alpha1-syntrophin, alpha1-, and Beta-dystrobrevins. We have established that Dp71ab forms a complex with Beta-Dystroglycan, alpha1-syntrophin, Beta-dystrobrevin, and alpha-, Beta- and gamma-sarcoglycans in undifferentiated PC12 cells. In differentiated PC12 cells, the complex composition changes since Dp71ab associates only with Beta-Dystroglycan, alpha1-syntrophin, Beta-dystrobrevin, and delta-sarcoglycan. Interestingly, neuronal nitric oxide synthase associates with the Dp71ab/DAPs complex during NGF treatment, raising the possibility that Dp71ab may be involved in signal transduction events during neuronal differentiation. J. Cell. Biochem. (c) 2007 Wiley-Liss, Inc.

Marius Sudol - One of the best experts on this subject based on the ideXlab platform.

  • Tyrosine phosphorylation of β-Dystroglycan at its WW domain binding motif, PPxY, recruits SH2 domain containing proteins
    Biochemistry, 2001
    Co-Authors: Federica Sotgia, Marius Sudol, Mark T Bedford, Tamara C. Petrucci, Hyangkyu Lee, Michael P. Lisanti
    Abstract:

    Beta-Dystroglycan is a ubiquitously expressed integral membrane protein that undergoes tyrosine phosphorylation in an adhesion-dependent manner. However, it remains unknown whether tyrosine-phosphorylated Beta-Dystroglycan interacts with SH2 domain containing proteins. Here, we show that the tyrosine phosphorylation of Beta-Dystroglycan is constitutively elevated in v-Src transformed cells. We next reconstituted this phosphorylation event in vivo by transiently coexpressing wild-type c-Src with a fusion protein containing full-length Beta-Dystroglycan. Our results demonstrate that Src-induced tyrosine phosphorylation of Beta-Dystroglycan is strictly dependent on the presence of a PPxY motif at its extreme C-terminus. In the nonphosphorylated state, this PPxY motif is normally recognized as a ligand by the WW domain; phosphorylation at this site blocks the binding of certain WW domain containing proteins. Using a GST fusion protein carrying the cytoplasmic tail of Beta-Dystroglycan, we identified five SH2 domain containing proteins that interact with Beta-Dystroglycan in a phosphorylation-dependent manner, including c-Src, Fyn, Csk, NCK, and SHC. We localized this binding activity to the PPxY motif by employing a panel of Beta-Dystroglycan-derived phosphopeptides. In addition, tyrosine phosphorylation of Beta-Dystroglycan in vivo resulted in the coimmunoprecipitation of the same SH2 domain containing proteins, and this binding event required the Beta-Dystroglycan C-terminal PPxY motif. We discuss the possibility that tyrosine phosphorylation of the PPxY motif within Beta-Dystroglycan may act as a regulatory switch to inhibit the binding of certain WW domain containing proteins, while recruiting SH2 domain containing proteins.

  • The interaction of dystrophin with β-Dystroglycan is regulated by tyrosine phosphorylation
    Cellular signalling, 2001
    Co-Authors: J.l. Ilsley, Marius Sudol, Steven J. Winder
    Abstract:

    Dystrophin and the dystrophin-associated protein complex (DAPC) have recently been implicated in cell signalling events. These proteins are ideally placed to transduce signals from the extracellular matrix (ECM) to the cytoskeleton. Here we show that Beta-Dystroglycan is tyrosine-phosphorylated in C2/C4 mouse myotubes. Tyrosine phosphorylation was detected by mobility shifts on SDS-polyacrylamide gels (SDS-PAGE) and confirmed by immunoprecipitation and two-dimensional gel electrophoresis. The potential functional significance of this tyrosine phosphorylation was investigated using peptide 'SPOTs' assays. Phosphorylation of tyrosine in the 15 most C-terminal amino acids of Beta-Dystroglycan disrupts its interaction with dystrophin. The tyrosine residue in Beta-Dystroglycan's WW-binding motif PPPY appears to be the most crucial in disrupting the Beta-Dystroglycan-dystrophin interaction. Beta-Dystroglycan forms the essential link between dystrophin and the rest of the DAPC. This regulation by tyrosine phosphorylation may have implications in the pathogenesis and treatment of Duchenne's muscular dystrophy (DMD).

  • Structure of a WW domain containing fragment of dystrophin in complex with Beta-Dystroglycan.
    Nature structural biology, 2000
    Co-Authors: Xin Huang, Marius Sudol, Andrzej Joachimiak, R. Zhang, Florence Poy, Michael J. Eck
    Abstract:

    Dystrophin and Beta-Dystroglycan are components of the dystrophin-glycoprotein complex (DGC), a multimolecular assembly that spans the cell membrane and links the actin cytoskeleton to the extracellular basal lamina. Defects in the dystrophin gene are the cause of Duchenne and Becker muscular dystrophies. The C-terminal region of dystrophin binds the cytoplasmic tail of Beta-Dystroglycan, in part through the interaction of its WW domain with a proline-rich motif in the tail of Beta-Dystroglycan. Here we report the crystal structure of this portion of dystrophin in complex with the proline-rich binding site in Beta-Dystroglycan. The structure shows that the dystrophin WW domain is embedded in an adjacent helical region that contains two EF-hand-like domains. The Beta-Dystroglycan peptide binds a composite surface formed by the WW domain and one of these EF-hands. Additionally, the structure reveals striking similarities in the mechanisms of proline recognition employed by WW domains and SH3 domains.

  • structure of a ww domain containing fragment of dystrophin in complex with Beta Dystroglycan
    Nature Structural & Molecular Biology, 2000
    Co-Authors: Xin Huang, Andrzej Joachimiak, R. Zhang, Marius Sudol
    Abstract:

    Dystrophin and β-Dystroglycan are components of the dystrophin–glycoprotein complex (DGC), a multimolecular assembly that spans the cell membrane and links the actin cytoskeleton to the extracellular basal lamina. Defects in the dystrophin gene are the cause of Duchenne and Becker muscular dystrophies. The C-terminal region of dystrophin binds the cytoplasmic tail of β-Dystroglycan, in part through the interaction of its WW domain with a proline-rich motif in the tail of β-Dystroglycan. Here we report the crystal structure of this portion of dystrophin in complex with the proline-rich binding site in β-Dystroglycan. The structure shows that the dystrophin WW domain is embedded in an adjacent helical region that contains two EF-hand-like domains. The β-Dystroglycan peptide binds a composite surface formed by the WW domain and one of these EF-hands. Additionally, the structure reveals striking similarities in the mechanisms of proline recognition employed by WW domains and SH3 domains.

  • Adhesion-dependent tyrosine phosphorylation of (Beta)-Dystroglycan regulates its interaction with utrophin.
    Journal of Cell Science, 2000
    Co-Authors: M. James, Marius Sudol, A. Nuttall, J.l. Ilsley, Katrin Ottersbach, J.m. Tinsley, Steven J. Winder
    Abstract:

    Many cell adhesion-dependent processes are regulated by tyrosine phosphorylation. In order to investigate the role of tyrosine phosphorylation of the utrophin-Dystroglycan complex we treated suspended or adherent cultures of HeLa cells with peroxyvanadate and immunoprecipitated (Beta)-Dystroglycan and utrophin from cell extracts. Western blotting of (β)-Dystroglycan and utrophin revealed adhesion- and peroxyvanadate-dependent mobility shifts which were recognised by anti-phospho-tyrosine antibodies. Using maltose binding protein fusion constructs to the carboxy-terminal domains of utrophin we were able to demonstrate specific interactions between the WW, EF and ZZ domains of utrophin and (Beta)-Dystroglycan by co-immunoprecipitation with endogenous (Beta)-Dystroglycan. In extracts from cells treated with peroxyvanadate, where endogenous (Beta)-Dystroglycan was tyrosine phosphorylated, (Beta)-Dystroglycan was no longer co-immunoprecipitated with utrophin fusion constructs. Peptide ‘SPOTs’ assays confirmed that tyrosine phosphorylation of (Beta)-Dystroglycan regulated the binding of utrophin. The phosphorylated tyrosine was identified as Y(892) in the (Beta)-Dystroglycan WW domain binding motif PPxY thus demonstrating the physiological regulation of the (Beta)-Dystroglycan/utrophin interaction by adhesion-dependent tyrosine phosphorylation.

Gérald Hugon - One of the best experts on this subject based on the ideXlab platform.

  • ZZ domain of dystrophin and utrophin: topology and mapping of a Beta-Dystroglycan interaction site.
    The Biochemical journal, 2007
    Co-Authors: Karim Hnia, Gérald Hugon, J. A. Fehrentz, A. Masmoudi, Dora Zouiten, Sonia Cantel, Delphine Chazalette, Ann Diment, Janice Bramham, Dominique Mornet
    Abstract:

    Dystrophin forms part of a vital link between actin cytoskeleton and extracellular matrix via the transmembrane adhesion receptor Dystroglycan. Dystrophin and its autosomal homologue utrophin interact with Beta-Dystroglycan via their highly conserved C-terminal cysteine-rich regions, comprising the WW domain (protein-protein interaction domain containing two conserved tryptophan residues), EF hand and ZZ domains. The EF hand region stabilizes the WW domain providing the main interaction site between dystrophin or utrophin and Dystroglycan. The ZZ domain, containing a predicted zinc finger motif, stabilizes the WW and EF hand domains and strengthens the overall interaction between dystrophin or utrophin and Beta-Dystroglycan. Using bacterially expressed ZZ domain, we demonstrate a conformational effect of zinc binding to the ZZ domain, and identify two zinc-binding regions within the ZZ domain by SPOTs overlay assays. Epitope mapping of the dystrophin ZZ domain was carried out with new monoclonal antibodies by ELISA, overlay assay and immunohistochemistry. One monoclonal antibody defined a discrete region of the ZZ domain that interacts with Beta-Dystroglycan. The epitope was localized to the conformationally sensitive second zinc-binding site in the ZZ domain. Our results suggest that residues 3326-3332 of dystrophin form a crucial part of the contact region between dystrophin and Beta-Dystroglycan and provide new insight into ZZ domain organization and function.

  • ZZ domain of dystrophin and utrophin: topology and mapping of a {Beta}-Dystroglycan interaction site
    Biochemical Journal, 2006
    Co-Authors: Karim Hnia, Gérald Hugon, J. A. Fehrentz, A. Masmoudi, Dora Zouiten, Sonia Cantel, Delphine Chazalette, Ann Diment, Janice Bramham, Dominique Mornet
    Abstract:

    Dystrophin forms part of a vital link between actin cytoskeleton and extracellular matrix via the transmembrane adhesion receptor Dystroglycan. Dystrophin and its autosomal homologue utrophin, interact with {Beta}-Dystroglycan via their highly conserved carboxy-terminal cysteine-rich regions, comprising WW, EF hand and ZZ domains. The EF hand region stabilises the WW domain providing the main interaction site between dystrophin or utrophin and Dystroglycan. The ZZ domain, containing a predicted zinc finger motif, stabilises the WW and EF hand domains and strengthens the overall interaction between dystrophin or utrophin and {Beta}-Dystroglycan. Using bacterially expressed ZZ domain we demonstrate a conformational effect of zinc binding to the ZZ domain, and identify two zinc binding regions within the ZZ domain by SPOTs overlay assays. Epitope-mapping of the dystrophin ZZ domain was carried out with new monoclonal antibodies by ELISA, overlay assay and immunohistochemistry. One monoclonal antibody defined a discrete region of the ZZ domain that interacts with {Beta}-Dystroglycan. The epitope was localised to the conformationaly sensitive second zinc binding site in the ZZ domain. Our results suggest that residues 3326-3332 of dystrophin, forms a crucial part of the contact region between dystrophin and {Beta}-Dystroglycan and provides new insight into ZZ domain organisation and function.

  • Effect of Beta-Dystroglycan processing on utrophin/Dp116 anchorage in normal and mdx mouse Schwann cell membrane.
    Neuroscience, 2006
    Co-Authors: K. Hnia, Gérald Hugon, François Rivier, A. Masmoudi, J. Mercier, Dominique Mornet
    Abstract:

    In the peripheral nervous system, utrophin and the short dystrophin isoform (Dp116) are co-localized at the outermost layer of the myelin sheath of nerve fibers; together with the Dystroglycan complex. Dp116 is associated with multiple glycoproteins, i.e. sarcoglycans, and alpha- and Beta-Dystroglycan, which anchor the cytoplasmic protein subcomplex to the extracellular basal lamina. In peripheral nerve, matrix metalloproteinase activity disrupts the Dystroglycan complex by cleaving the extracellular domain of Beta-Dystroglycan. Metalloproteinase creates a 30 kDa fragment of Beta-Dystroglycan, leading to a disruption of the link between the extracellular matrix and the cell membrane. Here we asked if the processing of the Beta-Dystroglycan could influence the anchorage of Dp116 and/or utrophin in normal and mdx Schwann cell membrane. We showed that metalloproteinase-9 was more activated in mdx nerve than in wild-type ones. This activation leads to an accumulation of the 30 kDa Beta-Dystroglycan isoform and has an impact on the anchorage of Dp116 and utrophin isoforms in mdx Schwann cells membrane. Our results showed that Dp116 had greater affinity to the full length form of Beta-Dystroglycan than the 30 kDa form. Moreover, we showed for the first time that the short isoform of utrophin (Up71) was over-expressed in mdx Schwann cells compared with wild-type. In addition, this utrophin isoform (Up71) seems to have greater affinity to the 30 kDa Beta-Dystroglycan which could explain the increased stabilization of this 30 kDa form at the membrane compartment. Our results highlight the potential participation of the short utrophin isoform and the cleaved form of Beta-Dystroglycan in mdx Schwann cell membrane architecture. We proposed that these two proteins could be implicated in Schwann cell proliferation in response to a microenvironment stress such as mediated by accumulating macrophages in mdx mouse muscle inflammation sites.

  • Pathological pattern of Mdx mice diaphragm correlates with gradual expression of the short utrophin isoform Up71.
    BBA - Biochimica et Biophysica Acta, 2006
    Co-Authors: Karim Hnia, Gérald Hugon, François Rivier, A. Masmoudi, Delphine Chazalette, Sylvie Tuffery-giraud, Marianne Vermaelen, Dominique Mornet
    Abstract:

    Utrophin gene is transcribed in a large mRNA of 13 kb that codes for a protein of 395 kDa. It shows amino acid identity with dystrophin of up to 73% and is widely expressed in muscle and non-muscle tissues. Up71 is a short utrophin product of the utrophin gene with the same cysteine-rich and C-terminal domains as full-length utrophin (Up395). Using RT-PCR, Western blots analysis, we demonstrated that Up71 is overexpressed in the mdx diaphragm, the most pathological muscle in dystrophin-deficient mdx mice, compared to wild-type C57BL/10 or other mdx skeletal muscles. Subsequently, we demonstrated that this isoform displayed an increased expression level up to 12 months, whereas full-length utrophin (Up395) decreased. In addition, Beta-Dystroglycan, the transmembrane glycoprotein that anchors the cytoplasmic C-terminal domain of utrophin, showed similar increase expression in mdx diaphragm, as opposed to other components of the dystrophin-associated protein complex (DAPC) such as alpha-dystrobrevin1 and alpha-sarcoglycan. We demonstrated that Up71 and Beta-Dystroglycan were progressively accumulated along the extrasynaptic region of regenerating clusters in mdx diaphragm. Our data provide novel functional insights into the pathological role of the Up71 isoform in dystrophinopathies.

  • effect of Beta Dystroglycan processing on utrophin dp116 anchorage in normal and mdx mouse schwann cell membrane
    Neuroscience, 2006
    Co-Authors: Karim Hnia, Gérald Hugon, François Rivier, A. Masmoudi, J. Mercier, Dominique Mornet
    Abstract:

    In the peripheral nervous system, utrophin and the short dystrophin isoform (Dp116) are co-localized at the outermost layer of the myelin sheath of nerve fibers; together with the Dystroglycan complex. Dp116 is associated with multiple glycoproteins, i.e. sarcoglycans, and alpha- and Beta-Dystroglycan, which anchor the cytoplasmic protein subcomplex to the extracellular basal lamina. In peripheral nerve, matrix metalloproteinase activity disrupts the Dystroglycan complex by cleaving the extracellular domain of Beta-Dystroglycan. Metalloproteinase creates a 30 kDa fragment of Beta-Dystroglycan, leading to a disruption of the link between the extracellular matrix and the cell membrane. Here we asked if the processing of the Beta-Dystroglycan could influence the anchorage of Dp116 and/or utrophin in normal and mdx Schwann cell membrane. We showed that metalloproteinase-9 was more activated in mdx nerve than in wild-type ones. This activation leads to an accumulation of the 30 kDa Beta-Dystroglycan isoform and has an impact on the anchorage of Dp116 and utrophin isoforms in mdx Schwann cells membrane. Our results showed that Dp116 had greater affinity to the full length form of Beta-Dystroglycan than the 30 kDa form. Moreover, we showed for the first time that the short isoform of utrophin (Up71) was over-expressed in mdx Schwann cells compared with wild-type. In addition, this utrophin isoform (Up71) seems to have greater affinity to the 30 kDa Beta-Dystroglycan which could explain the increased stabilization of this 30 kDa form at the membrane compartment. Our results highlight the potential participation of the short utrophin isoform and the cleaved form of Beta-Dystroglycan in mdx Schwann cell membrane architecture. We proposed that these two proteins could be implicated in Schwann cell proliferation in response to a microenvironment stress such as mediated by accumulating macrophages in mdx mouse muscle inflammation sites.

Karim Hnia - One of the best experts on this subject based on the ideXlab platform.

  • ZZ domain of dystrophin and utrophin: topology and mapping of a Beta-Dystroglycan interaction site.
    The Biochemical journal, 2007
    Co-Authors: Karim Hnia, Gérald Hugon, J. A. Fehrentz, A. Masmoudi, Dora Zouiten, Sonia Cantel, Delphine Chazalette, Ann Diment, Janice Bramham, Dominique Mornet
    Abstract:

    Dystrophin forms part of a vital link between actin cytoskeleton and extracellular matrix via the transmembrane adhesion receptor Dystroglycan. Dystrophin and its autosomal homologue utrophin interact with Beta-Dystroglycan via their highly conserved C-terminal cysteine-rich regions, comprising the WW domain (protein-protein interaction domain containing two conserved tryptophan residues), EF hand and ZZ domains. The EF hand region stabilizes the WW domain providing the main interaction site between dystrophin or utrophin and Dystroglycan. The ZZ domain, containing a predicted zinc finger motif, stabilizes the WW and EF hand domains and strengthens the overall interaction between dystrophin or utrophin and Beta-Dystroglycan. Using bacterially expressed ZZ domain, we demonstrate a conformational effect of zinc binding to the ZZ domain, and identify two zinc-binding regions within the ZZ domain by SPOTs overlay assays. Epitope mapping of the dystrophin ZZ domain was carried out with new monoclonal antibodies by ELISA, overlay assay and immunohistochemistry. One monoclonal antibody defined a discrete region of the ZZ domain that interacts with Beta-Dystroglycan. The epitope was localized to the conformationally sensitive second zinc-binding site in the ZZ domain. Our results suggest that residues 3326-3332 of dystrophin form a crucial part of the contact region between dystrophin and Beta-Dystroglycan and provide new insight into ZZ domain organization and function.

  • ZZ domain of dystrophin and utrophin: topology and mapping of a {Beta}-Dystroglycan interaction site
    Biochemical Journal, 2006
    Co-Authors: Karim Hnia, Gérald Hugon, J. A. Fehrentz, A. Masmoudi, Dora Zouiten, Sonia Cantel, Delphine Chazalette, Ann Diment, Janice Bramham, Dominique Mornet
    Abstract:

    Dystrophin forms part of a vital link between actin cytoskeleton and extracellular matrix via the transmembrane adhesion receptor Dystroglycan. Dystrophin and its autosomal homologue utrophin, interact with {Beta}-Dystroglycan via their highly conserved carboxy-terminal cysteine-rich regions, comprising WW, EF hand and ZZ domains. The EF hand region stabilises the WW domain providing the main interaction site between dystrophin or utrophin and Dystroglycan. The ZZ domain, containing a predicted zinc finger motif, stabilises the WW and EF hand domains and strengthens the overall interaction between dystrophin or utrophin and {Beta}-Dystroglycan. Using bacterially expressed ZZ domain we demonstrate a conformational effect of zinc binding to the ZZ domain, and identify two zinc binding regions within the ZZ domain by SPOTs overlay assays. Epitope-mapping of the dystrophin ZZ domain was carried out with new monoclonal antibodies by ELISA, overlay assay and immunohistochemistry. One monoclonal antibody defined a discrete region of the ZZ domain that interacts with {Beta}-Dystroglycan. The epitope was localised to the conformationaly sensitive second zinc binding site in the ZZ domain. Our results suggest that residues 3326-3332 of dystrophin, forms a crucial part of the contact region between dystrophin and {Beta}-Dystroglycan and provides new insight into ZZ domain organisation and function.

  • Pathological pattern of Mdx mice diaphragm correlates with gradual expression of the short utrophin isoform Up71.
    BBA - Biochimica et Biophysica Acta, 2006
    Co-Authors: Karim Hnia, Gérald Hugon, François Rivier, A. Masmoudi, Delphine Chazalette, Sylvie Tuffery-giraud, Marianne Vermaelen, Dominique Mornet
    Abstract:

    Utrophin gene is transcribed in a large mRNA of 13 kb that codes for a protein of 395 kDa. It shows amino acid identity with dystrophin of up to 73% and is widely expressed in muscle and non-muscle tissues. Up71 is a short utrophin product of the utrophin gene with the same cysteine-rich and C-terminal domains as full-length utrophin (Up395). Using RT-PCR, Western blots analysis, we demonstrated that Up71 is overexpressed in the mdx diaphragm, the most pathological muscle in dystrophin-deficient mdx mice, compared to wild-type C57BL/10 or other mdx skeletal muscles. Subsequently, we demonstrated that this isoform displayed an increased expression level up to 12 months, whereas full-length utrophin (Up395) decreased. In addition, Beta-Dystroglycan, the transmembrane glycoprotein that anchors the cytoplasmic C-terminal domain of utrophin, showed similar increase expression in mdx diaphragm, as opposed to other components of the dystrophin-associated protein complex (DAPC) such as alpha-dystrobrevin1 and alpha-sarcoglycan. We demonstrated that Up71 and Beta-Dystroglycan were progressively accumulated along the extrasynaptic region of regenerating clusters in mdx diaphragm. Our data provide novel functional insights into the pathological role of the Up71 isoform in dystrophinopathies.

  • interaction utrophine Beta Dystroglycan dans le muscle et le systeme nerveux peripherique de la souris deficiente en dystrophine
    2006
    Co-Authors: Karim Hnia
    Abstract:

    L'analyse des differentes interactions entre les membres du complexe associes a la dystrophine (DGC) a ete le centre de plusieurs etudes biochimiques. Le Beta-Dystroglycan prend une place primordiale au sein de ce complexe du fait de son role de proteine d'ancrage de la dystrophine et/ou de l'utrophine. De part son homologie structurale avec la dystrophine, l'utrophine est exprimee dans le muscle des patients DMD et de la souris modele de cette pathologie, la souris mdx. De plus en plus d'observations soulignent l'implication du Beta-Dystroglycan dans des evenements de signalisation. Comme l'organisation du complexe DGC, ces evenements sont perturbes dans le muscle deficient en dystrophine. L'interaction entre le Beta-Dystroglycan et l'utrophine est une association cle au niveau du sarcolemme de la fibre dystrophique. Nos travaux sont centres sur ce couple et en particulier sur differents processus et evenements qui influencent la stabilite de cette association. A la lumiere de nos resultats, confronte aux donnees de la litterature, nous avons montre que l'interaction entre l'utrophine et le Beta-Dystroglycan est fragilisee dans le muscle dystrophique. L'existence de l'isoform court de l'utrophine Up71 dans le diaphragme de la souris mdx ainsi que dans le nerf peripherique provoque une competition vis-a-vis de l'ancrage au Beta-Dystroglycan. D'autre part, l'affinite de l'utrophine pour le Beta-Dystroglycan est egalement fragilisee suite a l'activation du clivage du Beta-Dystroglycan dans le muscle et le nerf de la souris mdx par les metalloproteinases. L'activation de ces proteases est le resultat d'un enchainement de plusieurs cascades impliquant principalement certaines proteines de la cascade des MAP kinases et des proteines pro-inflammatoires. Des analyses structurales de l'association utrophine/ Beta-Dystroglycan, nous ont permis de mettre en evidence l'implication directe du motif ZZ de la region riche en cysteine de l'utrophine dans l'interaction avec le Beta-Dystroglycan et souligne que la fragilite de l'association l'utrophine/ Beta-Dystroglycan pourrait etre aussi le resultat d'une difference de la regulation de ce motif dans le muscle dystrophique.

  • effect of Beta Dystroglycan processing on utrophin dp116 anchorage in normal and mdx mouse schwann cell membrane
    Neuroscience, 2006
    Co-Authors: Karim Hnia, Gérald Hugon, François Rivier, A. Masmoudi, J. Mercier, Dominique Mornet
    Abstract:

    In the peripheral nervous system, utrophin and the short dystrophin isoform (Dp116) are co-localized at the outermost layer of the myelin sheath of nerve fibers; together with the Dystroglycan complex. Dp116 is associated with multiple glycoproteins, i.e. sarcoglycans, and alpha- and Beta-Dystroglycan, which anchor the cytoplasmic protein subcomplex to the extracellular basal lamina. In peripheral nerve, matrix metalloproteinase activity disrupts the Dystroglycan complex by cleaving the extracellular domain of Beta-Dystroglycan. Metalloproteinase creates a 30 kDa fragment of Beta-Dystroglycan, leading to a disruption of the link between the extracellular matrix and the cell membrane. Here we asked if the processing of the Beta-Dystroglycan could influence the anchorage of Dp116 and/or utrophin in normal and mdx Schwann cell membrane. We showed that metalloproteinase-9 was more activated in mdx nerve than in wild-type ones. This activation leads to an accumulation of the 30 kDa Beta-Dystroglycan isoform and has an impact on the anchorage of Dp116 and utrophin isoforms in mdx Schwann cells membrane. Our results showed that Dp116 had greater affinity to the full length form of Beta-Dystroglycan than the 30 kDa form. Moreover, we showed for the first time that the short isoform of utrophin (Up71) was over-expressed in mdx Schwann cells compared with wild-type. In addition, this utrophin isoform (Up71) seems to have greater affinity to the 30 kDa Beta-Dystroglycan which could explain the increased stabilization of this 30 kDa form at the membrane compartment. Our results highlight the potential participation of the short utrophin isoform and the cleaved form of Beta-Dystroglycan in mdx Schwann cell membrane architecture. We proposed that these two proteins could be implicated in Schwann cell proliferation in response to a microenvironment stress such as mediated by accumulating macrophages in mdx mouse muscle inflammation sites.

A. Masmoudi - One of the best experts on this subject based on the ideXlab platform.

  • ZZ domain of dystrophin and utrophin: topology and mapping of a Beta-Dystroglycan interaction site.
    The Biochemical journal, 2007
    Co-Authors: Karim Hnia, Gérald Hugon, J. A. Fehrentz, A. Masmoudi, Dora Zouiten, Sonia Cantel, Delphine Chazalette, Ann Diment, Janice Bramham, Dominique Mornet
    Abstract:

    Dystrophin forms part of a vital link between actin cytoskeleton and extracellular matrix via the transmembrane adhesion receptor Dystroglycan. Dystrophin and its autosomal homologue utrophin interact with Beta-Dystroglycan via their highly conserved C-terminal cysteine-rich regions, comprising the WW domain (protein-protein interaction domain containing two conserved tryptophan residues), EF hand and ZZ domains. The EF hand region stabilizes the WW domain providing the main interaction site between dystrophin or utrophin and Dystroglycan. The ZZ domain, containing a predicted zinc finger motif, stabilizes the WW and EF hand domains and strengthens the overall interaction between dystrophin or utrophin and Beta-Dystroglycan. Using bacterially expressed ZZ domain, we demonstrate a conformational effect of zinc binding to the ZZ domain, and identify two zinc-binding regions within the ZZ domain by SPOTs overlay assays. Epitope mapping of the dystrophin ZZ domain was carried out with new monoclonal antibodies by ELISA, overlay assay and immunohistochemistry. One monoclonal antibody defined a discrete region of the ZZ domain that interacts with Beta-Dystroglycan. The epitope was localized to the conformationally sensitive second zinc-binding site in the ZZ domain. Our results suggest that residues 3326-3332 of dystrophin form a crucial part of the contact region between dystrophin and Beta-Dystroglycan and provide new insight into ZZ domain organization and function.

  • ZZ domain of dystrophin and utrophin: topology and mapping of a {Beta}-Dystroglycan interaction site
    Biochemical Journal, 2006
    Co-Authors: Karim Hnia, Gérald Hugon, J. A. Fehrentz, A. Masmoudi, Dora Zouiten, Sonia Cantel, Delphine Chazalette, Ann Diment, Janice Bramham, Dominique Mornet
    Abstract:

    Dystrophin forms part of a vital link between actin cytoskeleton and extracellular matrix via the transmembrane adhesion receptor Dystroglycan. Dystrophin and its autosomal homologue utrophin, interact with {Beta}-Dystroglycan via their highly conserved carboxy-terminal cysteine-rich regions, comprising WW, EF hand and ZZ domains. The EF hand region stabilises the WW domain providing the main interaction site between dystrophin or utrophin and Dystroglycan. The ZZ domain, containing a predicted zinc finger motif, stabilises the WW and EF hand domains and strengthens the overall interaction between dystrophin or utrophin and {Beta}-Dystroglycan. Using bacterially expressed ZZ domain we demonstrate a conformational effect of zinc binding to the ZZ domain, and identify two zinc binding regions within the ZZ domain by SPOTs overlay assays. Epitope-mapping of the dystrophin ZZ domain was carried out with new monoclonal antibodies by ELISA, overlay assay and immunohistochemistry. One monoclonal antibody defined a discrete region of the ZZ domain that interacts with {Beta}-Dystroglycan. The epitope was localised to the conformationaly sensitive second zinc binding site in the ZZ domain. Our results suggest that residues 3326-3332 of dystrophin, forms a crucial part of the contact region between dystrophin and {Beta}-Dystroglycan and provides new insight into ZZ domain organisation and function.

  • Effect of Beta-Dystroglycan processing on utrophin/Dp116 anchorage in normal and mdx mouse Schwann cell membrane.
    Neuroscience, 2006
    Co-Authors: K. Hnia, Gérald Hugon, François Rivier, A. Masmoudi, J. Mercier, Dominique Mornet
    Abstract:

    In the peripheral nervous system, utrophin and the short dystrophin isoform (Dp116) are co-localized at the outermost layer of the myelin sheath of nerve fibers; together with the Dystroglycan complex. Dp116 is associated with multiple glycoproteins, i.e. sarcoglycans, and alpha- and Beta-Dystroglycan, which anchor the cytoplasmic protein subcomplex to the extracellular basal lamina. In peripheral nerve, matrix metalloproteinase activity disrupts the Dystroglycan complex by cleaving the extracellular domain of Beta-Dystroglycan. Metalloproteinase creates a 30 kDa fragment of Beta-Dystroglycan, leading to a disruption of the link between the extracellular matrix and the cell membrane. Here we asked if the processing of the Beta-Dystroglycan could influence the anchorage of Dp116 and/or utrophin in normal and mdx Schwann cell membrane. We showed that metalloproteinase-9 was more activated in mdx nerve than in wild-type ones. This activation leads to an accumulation of the 30 kDa Beta-Dystroglycan isoform and has an impact on the anchorage of Dp116 and utrophin isoforms in mdx Schwann cells membrane. Our results showed that Dp116 had greater affinity to the full length form of Beta-Dystroglycan than the 30 kDa form. Moreover, we showed for the first time that the short isoform of utrophin (Up71) was over-expressed in mdx Schwann cells compared with wild-type. In addition, this utrophin isoform (Up71) seems to have greater affinity to the 30 kDa Beta-Dystroglycan which could explain the increased stabilization of this 30 kDa form at the membrane compartment. Our results highlight the potential participation of the short utrophin isoform and the cleaved form of Beta-Dystroglycan in mdx Schwann cell membrane architecture. We proposed that these two proteins could be implicated in Schwann cell proliferation in response to a microenvironment stress such as mediated by accumulating macrophages in mdx mouse muscle inflammation sites.

  • Pathological pattern of Mdx mice diaphragm correlates with gradual expression of the short utrophin isoform Up71.
    BBA - Biochimica et Biophysica Acta, 2006
    Co-Authors: Karim Hnia, Gérald Hugon, François Rivier, A. Masmoudi, Delphine Chazalette, Sylvie Tuffery-giraud, Marianne Vermaelen, Dominique Mornet
    Abstract:

    Utrophin gene is transcribed in a large mRNA of 13 kb that codes for a protein of 395 kDa. It shows amino acid identity with dystrophin of up to 73% and is widely expressed in muscle and non-muscle tissues. Up71 is a short utrophin product of the utrophin gene with the same cysteine-rich and C-terminal domains as full-length utrophin (Up395). Using RT-PCR, Western blots analysis, we demonstrated that Up71 is overexpressed in the mdx diaphragm, the most pathological muscle in dystrophin-deficient mdx mice, compared to wild-type C57BL/10 or other mdx skeletal muscles. Subsequently, we demonstrated that this isoform displayed an increased expression level up to 12 months, whereas full-length utrophin (Up395) decreased. In addition, Beta-Dystroglycan, the transmembrane glycoprotein that anchors the cytoplasmic C-terminal domain of utrophin, showed similar increase expression in mdx diaphragm, as opposed to other components of the dystrophin-associated protein complex (DAPC) such as alpha-dystrobrevin1 and alpha-sarcoglycan. We demonstrated that Up71 and Beta-Dystroglycan were progressively accumulated along the extrasynaptic region of regenerating clusters in mdx diaphragm. Our data provide novel functional insights into the pathological role of the Up71 isoform in dystrophinopathies.

  • effect of Beta Dystroglycan processing on utrophin dp116 anchorage in normal and mdx mouse schwann cell membrane
    Neuroscience, 2006
    Co-Authors: Karim Hnia, Gérald Hugon, François Rivier, A. Masmoudi, J. Mercier, Dominique Mornet
    Abstract:

    In the peripheral nervous system, utrophin and the short dystrophin isoform (Dp116) are co-localized at the outermost layer of the myelin sheath of nerve fibers; together with the Dystroglycan complex. Dp116 is associated with multiple glycoproteins, i.e. sarcoglycans, and alpha- and Beta-Dystroglycan, which anchor the cytoplasmic protein subcomplex to the extracellular basal lamina. In peripheral nerve, matrix metalloproteinase activity disrupts the Dystroglycan complex by cleaving the extracellular domain of Beta-Dystroglycan. Metalloproteinase creates a 30 kDa fragment of Beta-Dystroglycan, leading to a disruption of the link between the extracellular matrix and the cell membrane. Here we asked if the processing of the Beta-Dystroglycan could influence the anchorage of Dp116 and/or utrophin in normal and mdx Schwann cell membrane. We showed that metalloproteinase-9 was more activated in mdx nerve than in wild-type ones. This activation leads to an accumulation of the 30 kDa Beta-Dystroglycan isoform and has an impact on the anchorage of Dp116 and utrophin isoforms in mdx Schwann cells membrane. Our results showed that Dp116 had greater affinity to the full length form of Beta-Dystroglycan than the 30 kDa form. Moreover, we showed for the first time that the short isoform of utrophin (Up71) was over-expressed in mdx Schwann cells compared with wild-type. In addition, this utrophin isoform (Up71) seems to have greater affinity to the 30 kDa Beta-Dystroglycan which could explain the increased stabilization of this 30 kDa form at the membrane compartment. Our results highlight the potential participation of the short utrophin isoform and the cleaved form of Beta-Dystroglycan in mdx Schwann cell membrane architecture. We proposed that these two proteins could be implicated in Schwann cell proliferation in response to a microenvironment stress such as mediated by accumulating macrophages in mdx mouse muscle inflammation sites.