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Katherine L Wilson - One of the best experts on this subject based on the ideXlab platform.
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The molecular basis of Emerin-Emerin and Emerin-BAF interactions
Journal of Cell Science, 2014Co-Authors: Jason M. Berk, Daniel Simon, Clifton R. Jenkins-houk, Jason W. Westerbeck, Line M. Grønning-wang, Cathrine R. Carlson, Katherine L WilsonAbstract:Emerin is a conserved membrane component of nuclear lamina structure. Here, we report an advance in understanding the molecular basis of Emerin function: intermolecular Emerin-Emerin association. There were two modes: one mediated by association of residues 170-220 in one Emerin molecule to residues 170-220 in another, and the second involving residues 170-220 and 1-132. Deletion analysis showed residues 187-220 contain a positive element essential for intermolecular association in cells. By contrast, deletion of residues 168-186 inactivated a proposed negative element, required to limit or control association. Association of GFP-Emerin with nuclear BAF in cells required the LEM domain (residues 1-47) and the positive element. Emerin peptide arrays revealed direct binding of residues 170-220 to residues 206-225 (the proposed positive element), residues 147-174 (particularly P(153)MYGRDSAYQSITHYRP(169)) and the LEM domain. Emerin residues 1-132 and 159-220 were each sufficient to bind lamin A or B1 tails in vitro, identifying two independent regions of molecular contact with lamins. These results, and predicted Emerin intrinsic disorder, support the hypothesis that there are multiple 'backbone' and LEM-domain configurations in a proposed intermolecular Emerin network at the nuclear envelope.
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o linked β n acetylglucosamine o glcnac regulates Emerin binding to barrier to autointegration factor baf in a chromatin and lamin b enriched niche
Journal of Biological Chemistry, 2013Co-Authors: Jason M. Berk, Sushmit Maitra, Andrew William Dawdy, Jeffrey Shabanowitz, Donald F Hunt, Katherine L WilsonAbstract:Abstract Emerin, a membrane component of nuclear “lamina” networks with lamins and barrier to autointegration factor (BAF), is highly O-GlcNAc-modified (“O-GlcNAcylated”) in mammalian cells. Mass spectrometry analysis revealed eight sites of O-GlcNAcylation, including Ser-53, Ser-54, Ser-87, Ser-171, and Ser-173. Emerin O-GlcNAcylation was reduced ∼50% by S53A or S54A mutation in vitro and in vivo. O-GlcNAcylation was reduced ∼66% by the triple S52A/S53A/S54A mutant, and S173A reduced O-GlcNAcylation of the S52A/S53A/S54A mutant by ∼30%, in vivo. We separated two populations of Emerin, A-type lamins and BAF; one population solubilized easily, and the other required sonication and included histones and B-type lamins. Emerin and BAF associated only in histone- and lamin-B-containing fractions. The S173D mutation specifically and selectively reduced GFP-Emerin association with BAF by 58% and also increased GFP-Emerin hyper-phosphorylation. We conclude that β-N-acetylglucosaminyltransferase, an essential enzyme, controls two regions in Emerin. The first region, defined by residues Ser-53 and Ser-54, flanks the LEM domain. O-GlcNAc modification at Ser-173, in the second region, is proposed to promote Emerin association with BAF in the chromatin/lamin B “niche.” These results reveal direct control of a conserved LEM domain nuclear lamina component by β-N-acetylglucosaminyltransferase, a nutrient sensor that regulates cell stress responses, mitosis, and epigenetics.
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Tyrosine phosphorylation of nuclear-membrane protein Emerin by Src, Abl and other kinases.
Journal of cell science, 2009Co-Authors: Kathryn E Tifft, Katherine A Bradbury, Katherine L WilsonAbstract:X-linked recessive Emery-Dreifuss muscular dystrophy (EDMD) is caused by loss of Emerin, a nuclear-membrane protein with roles in nuclear architecture, gene regulation and signaling. Phosphoproteomic studies have identified 13 sites of tyrosine phosphorylation in Emerin. We validated one study, confirming that Emerin is hyper-tyrosine-phosphorylated in Her2-overexpressing cells. We discovered that non-receptor tyrosine kinases Src and Abl each phosphorylate Emerin and a related protein, LAP2beta, directly. Src phosphorylated Emerin specifically at Y59, Y74 and Y95; the corresponding triple Y-to-F (;FFF') mutation reduced tyrosine phosphorylation by approximately 70% in vitro and in vivo. Substitutions that removed a single hydroxyl moiety either decreased (Y19F, Y34, Y161F) or increased (Y4F) Emerin binding to BAF in cells. Y19F, Y34F, Y161F and the FFF mutant also reduced recombinant Emerin binding to BAF from HeLa lysates, demonstrating the involvement of both LEM-domain and distal phosphorylatable tyrosines in binding BAF. We conclude that Emerin function is regulated by multiple tyrosine kinases, including Her2, Src and Abl, two of which (Her2, Src) regulate striated muscle. These findings suggest roles for Emerin as a downstream effector and ;signal integrator' for tyrosine kinase signaling pathway(s) at the nuclear envelope.
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Tyrosine phosphorylation of nuclear-membrane protein Emerin by Src, Abl and other kinases
Journal of Cell Science, 2009Co-Authors: Kathryn E Tifft, Katherine A Bradbury, Katherine L WilsonAbstract:X-linked recessive Emery-Dreifuss muscular dystrophy (EDMD) is caused by loss of Emerin, a nuclear-membrane protein with roles in nuclear architecture, gene regulation and signaling. Phosphoproteomic studies have identified 13 sites of tyrosine phosphorylation in Emerin. We validated one study, confirming that Emerin is hyper-tyrosine-phosphorylated in Her2-overexpressing cells. We discovered that non-receptor tyrosine kinases Src and Abl each phosphorylate Emerin and a related protein, LAP2β, directly. Src phosphorylated Emerin specifically at Y59, Y74 and Y95; the corresponding triple Y-to-F (`FFF9) mutation reduced tyrosine phosphorylation by ∼70% in vitro and in vivo. Substitutions that removed a single hydroxyl moiety either decreased (Y19F, Y34, Y161F) or increased (Y4F) Emerin binding to BAF in cells. Y19F, Y34F, Y161F and the FFF mutant also reduced recombinant Emerin binding to BAF from HeLa lysates, demonstrating the involvement of both LEM-domain and distal phosphorylatable tyrosines in binding BAF. We conclude that Emerin function is regulated by multiple tyrosine kinases, including Her2, Src and Abl, two of which (Her2, Src) regulate striated muscle. These findings suggest roles for Emerin as a downstream effector and `signal integrator9 for tyrosine kinase signaling pathway(s) at the nuclear envelope.
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An Emerin "proteome": purification of distinct Emerin-containing complexes from HeLa cells suggests molecular basis for diverse roles including gene regulation, mRNA splicing, signaling, mechanosensing, and nuclear architecture.
Biochemistry, 2007Co-Authors: James M. Holaska, Katherine L WilsonAbstract:Using recombinant bead-conjugated Emerin, we affinity-purified seven proteins from HeLa cell nuclear lysates that bind Emerin either directly or indirectly. These proteins were identified by mass spectrometry as nuclear alphaII-spectrin, nonmuscle myosin heavy chain alpha, Lmo7 (a predicted transcription regulator; reported separately), nuclear myosin I, beta-actin (reported separately), calponin 3, and SIKE. We now report that Emerin binds nuclear myosin I (NMI, a molecular motor) directly in vitro. Furthermore, bead-conjugated Emerin bound nuclear alphaII-spectrin and NMI equally well with or without ATP (which stimulates motor activity), whereas ATP decreased actin binding by 65%. Thus alphaII-spectrin and NMI interact stably with Emerin. To investigate the physiological relevance of these interactions, we used antibodies against Emerin to affinity-purify Emerin-associated protein complexes from HeLa cells and then further purified by ion-exchange chromatography to resolve by net charge and by size exclusion chromatography yielding six distinct Emerin-containing fractions (0.5-1.6 MDa). Western blotting suggested that each complex had distinct components involved in nuclear architecture (e.g., NMI, alphaII-spectrin, lamins) or gene or chromatin regulation (BAF, transcription regulators, HDACs). Additional constituents were identified by mass spectrometry. One putative gene-regulatory complex (complex 32) included core components of the nuclear corepressor (NCoR) complex, which mediates gene regulation by thyroid hormone and other nuclear receptors. When expressed in HeLa cells, FLAG-tagged NCoR subunits Gps2, HDAC3, TBLR1, and NCoR each co-immunoprecipitated Emerin, validating one putative complex. These findings support the hypothesis that Emerin scaffolds a variety of functionally distinct multiprotein complexes at the nuclear envelope in vivo. Notably included are nuclear myosin I-containing complexes that might sense and regulate mechanical tension at the nuclear envelope.
James M. Holaska - One of the best experts on this subject based on the ideXlab platform.
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Defects in Emerin-nucleoskeleton binding disrupt nuclear structure and promote breast cancer cell motility and metastasis.
Molecular cancer research : MCR, 2021Co-Authors: Alexandra G Liddane, Chelsea A Mcnamara, Mallory C Campbell, Isabelle Mercier, James M. HolaskaAbstract:Nuclear envelope proteins play an important role in regulating nuclear size and structure in cancer. Altered expression of nuclear lamins are found in many cancers and its expression is correlated with better clinical outcomes. The nucleus is the largest organelle in the cell with a diameter between 10 and 20 µm. Nuclear size significantly impacts cell migration. Nuclear structural changes are predicted to impact cancer metastasis by regulating cancer cell migration. Here we show Emerin regulates nuclear structure in invasive breast cancer cells to impact cancer metastasis. Invasive breast cancer cells had 40-50% less Emerin than control cells, which resulted in decreased nuclear size. Overexpression of GFP-Emerin in invasive breast cancer cells rescued nuclear size and inhibited migration through 3.0 and 8.0 µm pores. Mutational analysis showed Emerin binding to nucleoskeletal proteins was important for its regulation of nuclear structure, migration, and invasion. Importantly, Emerin expression inhibited lung metastasis by 91% in orthotopic mouse models of breast cancer. Emerin nucleoskeleton-binding mutants failed to inhibit metastasis. These results support a model whereby Emerin binding to the nucleoskeleton regulates nuclear structure to impact metastasis. In this model, Emerin plays a central role in metastatic transformation, since decreased Emerin expression during transformation causes the nuclear structural defects required for increased cell migration, intravasation and extravasation. Implications: Modulating Emerin expression and function represents new targets for therapeutic interventions of metastasis, since increased Emerin expression rescued cancer metastasis.
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The Molecular Basis and Biologic Significance of the β-Dystroglycan-Emerin Interaction
International journal of molecular sciences, 2020Co-Authors: Wendy Lilián Gómez-monsivais, Feliciano Monterrubio-ledezma, Jazmin Huerta-cantillo, Ricardo Mondragon-gonzalez, Alma Alamillo-iniesta, Ian García-aguirre, Paulina Margarita Azuara-medina, Raúl Argüello-garcía, Jhon Rivera-monroy, James M. HolaskaAbstract:β-dystroglycan (β-DG) assembles with lamins A/C and B1 and Emerin at the nuclear envelope (NE) to maintain proper nuclear architecture and function. To provide insight into the nuclear function of β-DG, we characterized the interaction between β-DG and Emerin at the molecular level. Emerin is a major NE protein that regulates multiple nuclear processes and whose deficiency results in Emery-Dreifuss muscular dystrophy (EDMD). Using truncated variants of β-DG and Emerin, via a series of in vitro and in vivo binding experiments and a tailored computational analysis, we determined that the β-DG-Emerin interaction is mediated at least in part by their respective transmembrane domains (TM). Using surface plasmon resonance assays we showed that Emerin binds to β-DG with high affinity (KD in the nanomolar range). Remarkably, the analysis of cells in which DG was knocked out demonstrated that loss of β-DG resulted in a decreased Emerin stability and impairment of Emerin-mediated processes. β-DG and Emerin are reciprocally required for their optimal targeting within the NE, as shown by immunofluorescence, western blotting and immunoprecipitation assays using Emerin variants with mutations in the TM domain and B-lymphocytes of a patient with EDMD. In summary, we demonstrated that β-DG plays a role as an Emerin interacting partner modulating its stability and function.
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EDMD-Causing Emerin Mutant Myogenic Progenitors Exhibit Impaired Differentiation Using Similar Mechanisms
Cells, 2020Co-Authors: Ashvin Iyer, James M. HolaskaAbstract:Mutations in the gene encoding Emerin (EMD) cause Emery-Dreifuss muscular dystrophy (EDMD1), an inherited disorder characterized by progressive skeletal muscle wasting, irregular heart rhythms and contractures of major tendons. The skeletal muscle defects seen in EDMD are caused by failure of muscle stem cells to differentiate and regenerate the damaged muscle. However, the underlying mechanisms remain poorly understood. Most EDMD1 patients harbor nonsense mutations and have no detectable Emerin protein. There are three EDMD-causing Emerin mutants (S54F, Q133H, and D95-99) that localize correctly to the nuclear envelope and are expressed at wildtype levels. We hypothesized these Emerin mutants would share in the disruption of key molecular pathways involved in myogenic differentiation. We generated myogenic progenitors expressing wildtype Emerin and each EDMD1-causing Emerin mutation (S54F, Q133H, D95-99) in an Emerin-null (EMD-/y) background. S54F, Q133H, and D95-99 failed to rescue EMD-/y myogenic differentiation, while wildtype Emerin efficiently rescued differentiation. RNA sequencing was done to identify pathways and networks important for Emerin regulation of myogenic differentiation. This analysis significantly reduced the number of pathways implicated in EDMD1 muscle pathogenesis.
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histone acetyltransferase inhibition rescues differentiation of Emerin deficient myogenic progenitors
Muscle & Nerve, 2020Co-Authors: Katherine A Bossone, Joseph A. Ellis, James M. HolaskaAbstract:Introduction Emery-Dreifuss muscular dystrophy (EDMD) is a disease characterized by skeletal muscle wasting, major tendon contractures, and cardiac conduction defects. Mutations in the gene encoding Emerin cause EDMD1. Our previous studies suggested that Emerin activation of histone deacetylase 3 (HDAC3) to reduce histone 4-lysine 5 (H4K5) acetylation (ac) is important for myogenic differentiation. Methods Pharmacological inhibitors (Nu9056, L002) of histone acetyltransferases targeting acetylated H4K5 were used to test whether increased acetylated H4K5 was responsible for the impaired differentiation seen in Emerin-deficient myogenic progenitors. Results Nu9056 and L002 rescued impaired differentiation in Emerin deficiency. SRT1720, which inhibits the nicotinamide adenine dinucleotide (NAD)+ -dependent deacetylase sirtuin 1 (SIRT1), failed to rescue myotube formation. Discussion We conclude that Emerin regulation of HDAC3 activity to affect H4K5 acetylation dynamics is important for myogenic differentiation. Targeting H4K5ac dynamics represents a potential new strategy for ameliorating the skeletal muscle wasting seen in EDMD1.
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Histone acetyltransferase inhibition rescues differentiation of Emerin‐deficient myogenic progenitors
Muscle & nerve, 2020Co-Authors: A B S Katherine Bossone, Joseph A. Ellis, James M. HolaskaAbstract:Introduction Emery-Dreifuss muscular dystrophy (EDMD) is a disease characterized by skeletal muscle wasting, major tendon contractures, and cardiac conduction defects. Mutations in the gene encoding Emerin cause EDMD1. Our previous studies suggested that Emerin activation of histone deacetylase 3 (HDAC3) to reduce histone 4-lysine 5 (H4K5) acetylation (ac) is important for myogenic differentiation. Methods Pharmacological inhibitors (Nu9056, L002) of histone acetyltransferases targeting acetylated H4K5 were used to test whether increased acetylated H4K5 was responsible for the impaired differentiation seen in Emerin-deficient myogenic progenitors. Results Nu9056 and L002 rescued impaired differentiation in Emerin deficiency. SRT1720, which inhibits the nicotinamide adenine dinucleotide (NAD)+ -dependent deacetylase sirtuin 1 (SIRT1), failed to rescue myotube formation. Discussion We conclude that Emerin regulation of HDAC3 activity to affect H4K5 acetylation dynamics is important for myogenic differentiation. Targeting H4K5ac dynamics represents a potential new strategy for ameliorating the skeletal muscle wasting seen in EDMD1.
Glenn E. Morris - One of the best experts on this subject based on the ideXlab platform.
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Effect of pathogenic mis-sense mutations in lamin A on its interaction with Emerin in vivo.
Journal of Cell Science, 2003Co-Authors: Ian Holt, Nguyen Thi Man, Cecilia Ostlund, Colin L Stewart, Howard J Worman, Glenn E. MorrisAbstract:Mutations in lamin A/C can cause Emery-Dreifuss muscular dystrophy (EDMD) or a related cardiomyopathy (CMD1A). Using transfection of lamin-A/C-deficient fibroblasts, we have studied the effects of nine pathogenic mutations on the ability of lamin A to assemble normally and to localize Emerin normally at the nuclear rim. Five mutations in the rod domain (L85R, N195K, E358K, M371K and R386K) affected the assembly of the lamina. With the exception of mutant L85R, all rod domain mutants induced the formation of large nucleoplasmic foci in about 10% of all nuclei. The presence of Emerin in these foci suggests that the interaction of lamin A with Emerin is not directly affected by the rod domain mutations. Three mutations in the tail region, R453W, W520S and R527P, might directly affect Emerin binding by disrupting the structure of the putative Emerin-binding site, because mutant lamin A localized normally to the nuclear rim but its ability to trap Emerin was impaired. Nucleoplasmic foci rarely formed in these three cases (
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effect of pathogenic mis sense mutations in lamin a on its interaction with Emerin in vivo
Journal of Cell Science, 2003Co-Authors: Ian Holt, Nguyen Thi Man, Cecilia Ostlund, Colin L Stewart, Howard J Worman, Glenn E. MorrisAbstract:Mutations in lamin A/C can cause Emery-Dreifuss muscular dystrophy (EDMD) or a related cardiomyopathy (CMD1A). Using transfection of lamin-A/C-deficient fibroblasts, we have studied the effects of nine pathogenic mutations on the ability of lamin A to assemble normally and to localize Emerin normally at the nuclear rim. Five mutations in the rod domain (L85R, N195K, E358K, M371K and R386K) affected the assembly of the lamina. With the exception of mutant L85R, all rod domain mutants induced the formation of large nucleoplasmic foci in about 10% of all nuclei. The presence of Emerin in these foci suggests that the interaction of lamin A with Emerin is not directly affected by the rod domain mutations. Three mutations in the tail region, R453W, W520S and R527P, might directly affect Emerin binding by disrupting the structure of the putative Emerin-binding site, because mutant lamin A localized normally to the nuclear rim but its ability to trap Emerin was impaired. Nucleoplasmic foci rarely formed in these three cases (<2%) but, when they did so, Emerin was absent, consistent with a direct effect of the mutations on Emerin binding. The lipodystrophy mutation R482Q, which causes a different phenotype and is believed to act through an Emerin-independent mechanism, was indistinguishable from wild-type in its localization and its ability to trap Emerin at the nuclear rim. The novel hypothesis suggested by the data is that EDMD/CMD1A mutations in the tail domain of lamin A/C work by direct impairment of Emerin interaction, whereas mutations in the rod region cause defective lamina assembly that might or might not impair Emerin capture at the nuclear rim. Subtle effects on the function of the lamina-Emerin complex in EDMD/CMD1A patients might be responsible for the skeletal and/or cardiac muscle phenotype.
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Emerin interacts in vitro with the splicing associated factor yt521 b
FEBS Journal, 2003Co-Authors: Fiona L. Wilkinson, James M. Holaska, Katherine L Wilson, S Manilal, Ian Holt, Zhayi Zhang, Aarti Sharma, Stefan Stamm, Glenn E. MorrisAbstract:Emerin is a nuclear membrane protein that interacts with lamin A/C at the nuclear envelope. Mutations in either Emerin or lamin A/C cause Emery–Dreifuss muscular dystrophy (EDMD). The functions of Emerin are poorly understood, but EDMD affects mainly skeletal and cardiac muscle. We used a high-stringency yeast two-hybrid method to screen a human heart cDNA library, with full-length Emerin as bait. Four out of five candidate interactors identified were nuclear proteins: lamin A, splicing factor YT521-B, proteasome subunit PA28γ and transcription factor vav-1. Specific binding between Emerin and the functional C-terminal domain of YT521-B was confirmed by pull-down assays and biomolecular interaction analysis (BIAcore). Inhibition by Emerin of YT521-B-dependent splice site selection in vivo suggests that the interaction is physiologically significant. A ‘bipartite’ binding site for YT521-B in Emerin was identified using alanine substitution or disease-associated mutations in Emerin. The transcription factor GCL (germ cell-less) has previously been shown to bind to the same site. The results are consistent with an emerging view that lamins and lamina-associated proteins, like Emerin, have a regulatory role, as well as a structural role in the nucleus. YT521-B joins a growing list of candidates for a role in a gene expression model of the pathogenesis of EDMD.
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Emerin interacts in vitro with the splicing‐associated factor, YT521‐B
European journal of biochemistry, 2003Co-Authors: Fiona L. Wilkinson, James M. Holaska, Katherine L Wilson, S Manilal, Ian Holt, Zhayi Zhang, Aarti Sharma, Stefan Stamm, Glenn E. MorrisAbstract:Emerin is a nuclear membrane protein that interacts with lamin A/C at the nuclear envelope. Mutations in either Emerin or lamin A/C cause Emery–Dreifuss muscular dystrophy (EDMD). The functions of Emerin are poorly understood, but EDMD affects mainly skeletal and cardiac muscle. We used a high-stringency yeast two-hybrid method to screen a human heart cDNA library, with full-length Emerin as bait. Four out of five candidate interactors identified were nuclear proteins: lamin A, splicing factor YT521-B, proteasome subunit PA28γ and transcription factor vav-1. Specific binding between Emerin and the functional C-terminal domain of YT521-B was confirmed by pull-down assays and biomolecular interaction analysis (BIAcore). Inhibition by Emerin of YT521-B-dependent splice site selection in vivo suggests that the interaction is physiologically significant. A ‘bipartite’ binding site for YT521-B in Emerin was identified using alanine substitution or disease-associated mutations in Emerin. The transcription factor GCL (germ cell-less) has previously been shown to bind to the same site. The results are consistent with an emerging view that lamins and lamina-associated proteins, like Emerin, have a regulatory role, as well as a structural role in the nucleus. YT521-B joins a growing list of candidates for a role in a gene expression model of the pathogenesis of EDMD.
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How does a g993t mutation in the Emerin gene cause Emery-Dreifuss muscular dystrophy?
Biochemical and biophysical research communications, 2001Co-Authors: Ian Holt, S Manilal, Lisa Clements, Glenn E. MorrisAbstract:X-linked Emery-Dreifuss muscular dystrophy is usually caused by absence of the nuclear membrane protein, Emerin, due to nonsense mutations or deletions, but a few missense mutations also exist. A pathogenic g993t mutation causes a Q133H change in the nuclear targeting region of Emerin, but it may also reduce Emerin levels by affecting mRNA splicing. We have introduced the g993t mutation by in vitro mutagenesis and studied the effect of Q133H on nuclear targeting by transfection of COS-7 cells. No qualitative or quantitative differences in nuclear targeting were observed between normal and mutant Emerin. Quantitative BIAcore analysis showed no significant change in lamin A binding to Emerin when the mutation was present. We conclude that Q133 is not essential for nuclear targeting of Emerin or its interaction with lamin A. Reduced Emerin levels due to altered splicing or defective interaction with an unidentified binding partner remain possible pathogenic mechanisms.
Ian Holt - One of the best experts on this subject based on the ideXlab platform.
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effect of pathogenic mis sense mutations in lamin a on its interaction with Emerin in vivo
Journal of Cell Science, 2003Co-Authors: Ian Holt, Nguyen Thi Man, Cecilia Ostlund, Colin L Stewart, Howard J Worman, Glenn E. MorrisAbstract:Mutations in lamin A/C can cause Emery-Dreifuss muscular dystrophy (EDMD) or a related cardiomyopathy (CMD1A). Using transfection of lamin-A/C-deficient fibroblasts, we have studied the effects of nine pathogenic mutations on the ability of lamin A to assemble normally and to localize Emerin normally at the nuclear rim. Five mutations in the rod domain (L85R, N195K, E358K, M371K and R386K) affected the assembly of the lamina. With the exception of mutant L85R, all rod domain mutants induced the formation of large nucleoplasmic foci in about 10% of all nuclei. The presence of Emerin in these foci suggests that the interaction of lamin A with Emerin is not directly affected by the rod domain mutations. Three mutations in the tail region, R453W, W520S and R527P, might directly affect Emerin binding by disrupting the structure of the putative Emerin-binding site, because mutant lamin A localized normally to the nuclear rim but its ability to trap Emerin was impaired. Nucleoplasmic foci rarely formed in these three cases (<2%) but, when they did so, Emerin was absent, consistent with a direct effect of the mutations on Emerin binding. The lipodystrophy mutation R482Q, which causes a different phenotype and is believed to act through an Emerin-independent mechanism, was indistinguishable from wild-type in its localization and its ability to trap Emerin at the nuclear rim. The novel hypothesis suggested by the data is that EDMD/CMD1A mutations in the tail domain of lamin A/C work by direct impairment of Emerin interaction, whereas mutations in the rod region cause defective lamina assembly that might or might not impair Emerin capture at the nuclear rim. Subtle effects on the function of the lamina-Emerin complex in EDMD/CMD1A patients might be responsible for the skeletal and/or cardiac muscle phenotype.
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Effect of pathogenic mis-sense mutations in lamin A on its interaction with Emerin in vivo.
Journal of Cell Science, 2003Co-Authors: Ian Holt, Nguyen Thi Man, Cecilia Ostlund, Colin L Stewart, Howard J Worman, Glenn E. MorrisAbstract:Mutations in lamin A/C can cause Emery-Dreifuss muscular dystrophy (EDMD) or a related cardiomyopathy (CMD1A). Using transfection of lamin-A/C-deficient fibroblasts, we have studied the effects of nine pathogenic mutations on the ability of lamin A to assemble normally and to localize Emerin normally at the nuclear rim. Five mutations in the rod domain (L85R, N195K, E358K, M371K and R386K) affected the assembly of the lamina. With the exception of mutant L85R, all rod domain mutants induced the formation of large nucleoplasmic foci in about 10% of all nuclei. The presence of Emerin in these foci suggests that the interaction of lamin A with Emerin is not directly affected by the rod domain mutations. Three mutations in the tail region, R453W, W520S and R527P, might directly affect Emerin binding by disrupting the structure of the putative Emerin-binding site, because mutant lamin A localized normally to the nuclear rim but its ability to trap Emerin was impaired. Nucleoplasmic foci rarely formed in these three cases (
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Emerin interacts in vitro with the splicing associated factor yt521 b
FEBS Journal, 2003Co-Authors: Fiona L. Wilkinson, James M. Holaska, Katherine L Wilson, S Manilal, Ian Holt, Zhayi Zhang, Aarti Sharma, Stefan Stamm, Glenn E. MorrisAbstract:Emerin is a nuclear membrane protein that interacts with lamin A/C at the nuclear envelope. Mutations in either Emerin or lamin A/C cause Emery–Dreifuss muscular dystrophy (EDMD). The functions of Emerin are poorly understood, but EDMD affects mainly skeletal and cardiac muscle. We used a high-stringency yeast two-hybrid method to screen a human heart cDNA library, with full-length Emerin as bait. Four out of five candidate interactors identified were nuclear proteins: lamin A, splicing factor YT521-B, proteasome subunit PA28γ and transcription factor vav-1. Specific binding between Emerin and the functional C-terminal domain of YT521-B was confirmed by pull-down assays and biomolecular interaction analysis (BIAcore). Inhibition by Emerin of YT521-B-dependent splice site selection in vivo suggests that the interaction is physiologically significant. A ‘bipartite’ binding site for YT521-B in Emerin was identified using alanine substitution or disease-associated mutations in Emerin. The transcription factor GCL (germ cell-less) has previously been shown to bind to the same site. The results are consistent with an emerging view that lamins and lamina-associated proteins, like Emerin, have a regulatory role, as well as a structural role in the nucleus. YT521-B joins a growing list of candidates for a role in a gene expression model of the pathogenesis of EDMD.
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Emerin interacts in vitro with the splicing‐associated factor, YT521‐B
European journal of biochemistry, 2003Co-Authors: Fiona L. Wilkinson, James M. Holaska, Katherine L Wilson, S Manilal, Ian Holt, Zhayi Zhang, Aarti Sharma, Stefan Stamm, Glenn E. MorrisAbstract:Emerin is a nuclear membrane protein that interacts with lamin A/C at the nuclear envelope. Mutations in either Emerin or lamin A/C cause Emery–Dreifuss muscular dystrophy (EDMD). The functions of Emerin are poorly understood, but EDMD affects mainly skeletal and cardiac muscle. We used a high-stringency yeast two-hybrid method to screen a human heart cDNA library, with full-length Emerin as bait. Four out of five candidate interactors identified were nuclear proteins: lamin A, splicing factor YT521-B, proteasome subunit PA28γ and transcription factor vav-1. Specific binding between Emerin and the functional C-terminal domain of YT521-B was confirmed by pull-down assays and biomolecular interaction analysis (BIAcore). Inhibition by Emerin of YT521-B-dependent splice site selection in vivo suggests that the interaction is physiologically significant. A ‘bipartite’ binding site for YT521-B in Emerin was identified using alanine substitution or disease-associated mutations in Emerin. The transcription factor GCL (germ cell-less) has previously been shown to bind to the same site. The results are consistent with an emerging view that lamins and lamina-associated proteins, like Emerin, have a regulatory role, as well as a structural role in the nucleus. YT521-B joins a growing list of candidates for a role in a gene expression model of the pathogenesis of EDMD.
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How does a g993t mutation in the Emerin gene cause Emery-Dreifuss muscular dystrophy?
Biochemical and biophysical research communications, 2001Co-Authors: Ian Holt, S Manilal, Lisa Clements, Glenn E. MorrisAbstract:X-linked Emery-Dreifuss muscular dystrophy is usually caused by absence of the nuclear membrane protein, Emerin, due to nonsense mutations or deletions, but a few missense mutations also exist. A pathogenic g993t mutation causes a Q133H change in the nuclear targeting region of Emerin, but it may also reduce Emerin levels by affecting mRNA splicing. We have introduced the g993t mutation by in vitro mutagenesis and studied the effect of Q133H on nuclear targeting by transfection of COS-7 cells. No qualitative or quantitative differences in nuclear targeting were observed between normal and mutant Emerin. Quantitative BIAcore analysis showed no significant change in lamin A binding to Emerin when the mutation was present. We conclude that Q133 is not essential for nuclear targeting of Emerin or its interaction with lamin A. Reduced Emerin levels due to altered splicing or defective interaction with an unidentified binding partner remain possible pathogenic mechanisms.
Howard J Worman - One of the best experts on this subject based on the ideXlab platform.
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Muscular Dystrophy Mutations Impair the Nuclear Envelope Emerin Self-assembly Properties.
ACS Chemical Biology, 2015Co-Authors: Isaline Herrada, Camille Samson, Cecilia Ostlund, Howard J Worman, Christophe Velours, Louis Renault, Pierre Chervy, Dmytro Puchkov, Brigitte Buendia, Sophie Zinn-justinAbstract:More than 100 genetic mutations causing X-linked Emery-Dreifuss muscular dystrophy have been identified in the gene encoding the integral inner nuclear membrane protein Emerin. Most mutations are nonsense or frameshift mutations that lead to the absence of Emerin in cells. Only very few cases are due to missense or short in-frame deletions. Molecular mechanisms explaining the corresponding Emerin variants' loss of function are particularly difficult to identify because of the mostly intrinsically disordered state of the Emerin nucleoplasmic region. We now demonstrate that this EmN region can be produced as a disordered monomer, as revealed by nuclear magnetic resonance, but rapidly self-assembles in vitro. Increases in concentration and temperature favor the formation of long curvilinear filaments with diameters of approximately 10 nm, as observed by electron microscopy. Assembly of these filaments can be followed by fluorescence through Thioflavin-T binding and by Fourier-transform Infrared spectrometry through formation of β-structures. Analysis of the assembly properties of five EmN variants reveals that del95-99 and Q133H impact filament assembly capacities. In cells, these variants are located at the nuclear envelope, but the corresponding quantities of Emerin-Emerin and Emerin-lamin proximities are decreased compared to wild-type protein. Furthermore, variant P183H favors EmN aggregation in vitro, and variant P183T provokes Emerin accumulation in cytoplasmic foci in cells. Substitution of residue Pro183 might systematically favor oligomerization, leading to Emerin aggregation and mislocalization in cells. Our results suggest that Emerin self-assembly is necessary for its proper function and that a loss of either the protein itself or its ability to self-assemble causes muscular dystrophy.
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Emerin Organizes Actin Flow for Nuclear Movement and Centrosome Orientation in Migrating Fibroblasts
Molecular biology of the cell, 2013Co-Authors: Wakam Chang, Howard J Worman, Eric S. Folker, Gregg G. GundersenAbstract:In migrating fibroblasts, rearward movement of the nucleus orients the centrosome toward the leading edge. Nuclear movement results from coupling rearward-moving, dorsal actin cables to the nucleus by linear arrays of nesprin-2G and SUN2, termed transmembrane actin-associated nuclear (TAN) lines. A-type lamins anchor TAN lines, prompting us to test whether Emerin, a nuclear membrane protein that interacts with lamins and TAN line proteins, contributes to nuclear movement. In fibroblasts depleted of Emerin, nuclei moved nondirectionally or completely failed to move. Consistent with these nuclear movement defects, dorsal actin cable flow was nondirectional in cells lacking Emerin. TAN lines formed normally in cells lacking Emerin and were coordinated with the erratic nuclear movements, although in 20% of the cases, TAN lines slipped over immobile nuclei. Myosin II drives actin flow, and depletion of myosin IIB, but not myosin IIA, showed similar nondirectional nuclear movement and actin flow as in Emerin-depleted cells. Myosin IIB specifically coimmunoprecipitated with Emerin, and Emerin depletion prevented myosin IIB localization near nuclei. These results show that Emerin functions with myosin IIB to polarize actin flow and nuclear movement in fibroblasts, suggesting a novel function for the nuclear envelope in organizing directional actin flow and cytoplasmic polarity.
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Lamina-associated polypeptide-1 interacts with the muscular dystrophy protein Emerin and is essential for skeletal muscle maintenance.
Developmental cell, 2013Co-Authors: Ji-yeon Shin, Howard J Worman, Iván Méndez-lópez, Yuexia Wang, Arthur P. Hays, Kurenai Tanji, Jay H. Lefkowitch, P. Christian Schulze, William T. DauerAbstract:X-linked Emery-Dreifuss muscular dystrophy is caused by loss of function of Emerin, an integral protein of the inner nuclear membrane. Yet Emerin null mice are essentially normal, suggesting the existence of a critical compensating factor. We show that the lamina-associated polypeptide1 (LAP1) interacts with Emerin. Conditional deletion of LAP1 from striated muscle causes muscular dystrophy; this pathology is worsened in the absence of Emerin. LAP1 levels are significantly higher in mouse than human skeletal muscle, and reducing LAP1 by approximately half in mice also induces muscle abnormalities in Emerin null mice. Conditional deletion of LAP1 from hepatocytes yields mice that exhibit normal liver function and are indistinguishable from littermate controls. These results establish that LAP1 interacts physically and functionally with Emerin and plays an essential and selective role in skeletal muscle maintenance. They also highlight how dissecting differences between mouse and human phenotypes can provide fundamental insights into disease mechanisms.
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Reduced expression of A-type lamins and Emerin activates extracellular signal-regulated kinase in cultured cells.
Biochimica et biophysica acta, 2008Co-Authors: Antoine Muchir, Howard J WormanAbstract:Abstract Background: Mutations in genes encoding A-type lamins and Emerin cause cardiomyopathy and muscular dystrophy. We previously showed activation of the extracellular signal-regulated kinase (ERK) branch of the mitogen-activated protein kinase (MAPK) cascade in hearts of mice with mutations in these genes. Here, we tested the hypothesis that reducing A-type lamins and Emerin in cultured cells activate ERK signaling. Methods: We used siRNA to knockdown A-type lamins and Emerin in HeLa and C2C12 cells. Activation of ERK was assessed by immunoblotting and immunofluorescence microscopy with antibodies against phosphorylated protein and by using real-time RT-PCR to measure RNAs encoded by genes for transcription factors stimulated by ERK. Results: Knockdown of A-type lamins and Emerin in HeLa and C2C12 stimulated phosphorylation and nuclear translocation of ERK as well as activation of genes encoding downstream transcription factors. A MAPK/ERK kinase (MEK) inhibitor reduced ERK phosphorylation in cells with reduced expression of A-type lamins and Emerin. Conclusions: These results provide proof for the hypothesis that altered expression of Emerin and A-type lamins activates ERK signaling, which in turn can cause cardiomyopathy. General significance : ERK is a potential target for the pharmacological treatment of cardiomyopathy caused by mutations in the genes encoding Emerin and A-type lamins.
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effect of pathogenic mis sense mutations in lamin a on its interaction with Emerin in vivo
Journal of Cell Science, 2003Co-Authors: Ian Holt, Nguyen Thi Man, Cecilia Ostlund, Colin L Stewart, Howard J Worman, Glenn E. MorrisAbstract:Mutations in lamin A/C can cause Emery-Dreifuss muscular dystrophy (EDMD) or a related cardiomyopathy (CMD1A). Using transfection of lamin-A/C-deficient fibroblasts, we have studied the effects of nine pathogenic mutations on the ability of lamin A to assemble normally and to localize Emerin normally at the nuclear rim. Five mutations in the rod domain (L85R, N195K, E358K, M371K and R386K) affected the assembly of the lamina. With the exception of mutant L85R, all rod domain mutants induced the formation of large nucleoplasmic foci in about 10% of all nuclei. The presence of Emerin in these foci suggests that the interaction of lamin A with Emerin is not directly affected by the rod domain mutations. Three mutations in the tail region, R453W, W520S and R527P, might directly affect Emerin binding by disrupting the structure of the putative Emerin-binding site, because mutant lamin A localized normally to the nuclear rim but its ability to trap Emerin was impaired. Nucleoplasmic foci rarely formed in these three cases (<2%) but, when they did so, Emerin was absent, consistent with a direct effect of the mutations on Emerin binding. The lipodystrophy mutation R482Q, which causes a different phenotype and is believed to act through an Emerin-independent mechanism, was indistinguishable from wild-type in its localization and its ability to trap Emerin at the nuclear rim. The novel hypothesis suggested by the data is that EDMD/CMD1A mutations in the tail domain of lamin A/C work by direct impairment of Emerin interaction, whereas mutations in the rod region cause defective lamina assembly that might or might not impair Emerin capture at the nuclear rim. Subtle effects on the function of the lamina-Emerin complex in EDMD/CMD1A patients might be responsible for the skeletal and/or cardiac muscle phenotype.