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Michel Labouesse - One of the best experts on this subject based on the ideXlab platform.
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crt 1 calreticulin and the e3 ligase eel 1 huwe1 control hemidesmosome maturation in c elegans development
Current Biology, 2010Co-Authors: Hala Zahreddine, Marie Diogon, Yasuko Nagamatsu, Huimin Zhang, Michel LabouesseAbstract:Summary Hemidesmosomes connect the extracellular matrix (ECM) to intermediate filaments through ECM receptors and Plakins (plectin and BPAG1e). They affect tissue integrity, wound healing, and carcinoma invasion [1]. Although biochemical and time-lapse studies indicate that α6β4-integrin (ECM receptor) and plectin play a central role in modulating hemidesmosome disassembly [2–5], the mechanisms controlling hemidesmosome biogenesis in vivo remain poorly understood. The nematode C. elegans provides a powerful genetic model to address this issue. We performed a genome-wide RNA interference screen in C. elegans , searching for genes that decrease the viability of a weak VAB-10A/Plakin mutant. We identified 14 genes that have human homologs with predicted roles in different cellular processes. We further characterized two genes encoding the chaperone CRT-1/calreticulin and the HECT domain E3 ubiquitin ligase EEL-1/HUWE1. CRT-1 controls by as little as 2-fold the abundance of UNC-52/perlecan, an essential hemidesmosome ECM ligand. Likewise, EEL-1 fine tunes by 2-fold the abundance of myotactin, the putative hemidesmosome ECM receptor. CRT-1 and EEL-1 activities, and by extension other genes identified in our screen, are essential during embryonic development to enable hemidesmosomes exposed to mechanical tension to mature into a tension-resistant form. Our findings should help understand how hemidesmosome dynamics are regulated in vertebrate systems.
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CRT-1/calreticulin and the E3 ligase EEL-1/HUWE1 control hemidesmosome maturation in C. elegans development.
Current Biology - CB, 2010Co-Authors: Hala Zahreddine, Marie Diogon, Yasuko Nagamatsu, Huimin Zhang, Michel LabouesseAbstract:Hemidesmosomes connect the extracellular matrix (ECM) to intermediate filaments through ECM receptors and Plakins (plectin and BPAG1e). They affect tissue integrity, wound healing, and carcinoma invasion. Although biochemical and time-lapse studies indicate that alpha6beta4-integrin (ECM receptor) and plectin play a central role in modulating hemidesmosome disassembly, the mechanisms controlling hemidesmosome biogenesis in vivo remain poorly understood. The nematode C. elegans provides a powerful genetic model to address this issue. We performed a genome-wide RNA interference screen in C. elegans, searching for genes that decrease the viability of a weak VAB-10A/Plakin mutant. We identified 14 genes that have human homologs with predicted roles in different cellular processes. We further characterized two genes encoding the chaperone CRT-1/calreticulin and the HECT domain E3 ubiquitin ligase EEL-1/HUWE1. CRT-1 controls by as little as 2-fold the abundance of UNC-52/perlecan, an essential hemidesmosome ECM ligand. Likewise, EEL-1 fine tunes by 2-fold the abundance of myotactin, the putative hemidesmosome ECM receptor. CRT-1 and EEL-1 activities, and by extension other genes identified in our screen, are essential during embryonic development to enable hemidesmosomes exposed to mechanical tension to mature into a tension-resistant form. Our findings should help understand how hemidesmosome dynamics are regulated in vertebrate systems.
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The Caenorhabditis elegans vab-10 spectraPlakin isoforms protect the epidermis against internal and external forces
The Journal of cell biology, 2003Co-Authors: Julia M. Bosher, Bum-soo Hahn, Renaud Legouis, Satis Sookhareea, Robby M. Weimer, Anne Gansmuller, Andrew D. Chisholm, Ann M. Rose, Jean-louis Bessereau, Michel LabouesseAbstract:Morphogenesis of the Caenorhabditis elegans embryo is driven by actin microfilaments in the epidermis and by sarcomeres in body wall muscles. Both tissues are mechanically coupled, most likely through specialized attachment structures called fibrous organelles (FOs) that connect muscles to the cuticle across the epidermis. Here, we report the identification of new mutations in a gene known as vab-10, which lead to severe morphogenesis defects, and show that vab-10 corresponds to the C. elegans spectraPlakin locus. Our analysis of vab-10 reveals novel insights into the role of this Plakin subfamily. vab-10 generates isoforms related either to plectin (termed VAB-10A) or to microtubule actin cross-linking factor Plakins (termed VAB-10B). Using specific antibodies and mutations, we show that VAB-10A and VAB-10B have distinct distributions and functions in the epidermis. Loss of VAB-10A impairs the integrity of FOs, leading to epidermal detachment from the cuticle and muscles, hence demonstrating that FOs are functionally and molecularly related to hemidesmosomes. We suggest that this isoform protects against forces external to the epidermis. In contrast, lack of VAB-10B leads to increased epidermal thickness during embryonic morphogenesis when epidermal cells change shape. We suggest that this isoform protects cells against tension that builds up within the epidermis.
Ronald K.h. Liem - One of the best experts on this subject based on the ideXlab platform.
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Plakins in development and disease
Experimental Cell Research, 2007Co-Authors: Arnoud Sonnenberg, Ronald K.h. LiemAbstract:Abstract Plakins are large multi-domain molecules that have various functions to link cytoskeletal elements together and to connect them to junctional complexes. Plakins were first identified in epithelial cells where they were found to connect the intermediate filaments to desmosomes and hemidesmosomes [Ruhrberg, C., and Watt, F.M. (1997). The Plakin family: versatile organizers of cytoskeletal architecture. Curr Opin Genet Dev 7, 392–397.]. They were subsequently found to be important for the integrity of muscle cells. Most recently, they have been found in the nervous system, where their functions appear to be more complex, including cross-linking of microtubules (MTs) and actin filaments [Leung, C.L., Zheng, M., Prater, S.M., and Liem, R.K. (2001). The BPAG1 locus: Alternative splicing produces multiple isoforms with distinct cytoskeletal linker domains, including predominant isoforms in neurons and muscles. J Cell Biol 154, 691–697., Leung, C.L., Sun, D., Zheng, M., Knowles, D.R., and Liem, R.K. (1999). Microtubule actin cross-linking factor (MACF): a hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons. J Cell Biol 147, 1275–1286.]. These Plakins have also indicated their relationship to the spectrin superfamily of proteins and the Plakins appear to be evolutionarily related to the spectrins, but have diverged to perform different specialized functions. In invertebrates, a single Plakin is present in both Drosophila melanogaster and Caenorhabditis elegans, which resemble the more complex Plakins found in mammals [Roper, K., Gregory, S.L., and Brown, N.H. (2002). The ‘spectraPlakins’: cytoskeletal giants with characteristics of both spectrin and Plakin families. J Cell Sci 115, 4215–4225.]. In contrast, there are seven Plakins found in mammals and most of them have alternatively spliced forms leading to a very complex group of proteins with potential tissue specific functions [Jefferson, J.J., Leung, C.L., and Liem, R.K. (2004). Plakins: goliaths that link cell junctions and the cytoskeleton. Nat Rev Mol Cell Biol 5, 542–553.]. In this review, we will first describe the Plakins, desmoPlakin, plectin, envoPlakin and periPlakin and then describe two other mammalian Plakins, Bullous pemphigoid antigen 1 (BPAG1) and microtubule actin cross-linking factor 1 (MACF1), that are expressed in multiple isoforms in different tissues. We will also describe the relationship of these two proteins to the invertebrate Plakins, shortstop (shot) in Drosophila and VAB-10 in C. elegans. Finally, we will describe an unusual mammalian Plakin, called epiPlakin.
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Structural analysis of the Plakin domain of bullous pemphigoid antigen1 (BPAG1) suggests that Plakins are members of the spectrin superfamily.
Journal of molecular biology, 2006Co-Authors: Julius J. Jefferson, Carlo Ciatto, Lawrence Shapiro, Ronald K.h. LiemAbstract:Bullous pemphigoid antigen 1 (BPAG1) is a member of the Plakin family of proteins. The Plakins are multi-domain proteins that have been shown to interact with microtubules, actin filaments and intermediate filaments, as well as proteins found in cellular junctions. These interactions are mediated through different domains on the Plakins. The interactions between Plakins and components of specialized cell junctions such as desmosomes and hemidesmosomes are mediated through the so-called Plakin domain, which is a common feature of the Plakins. We report the crystal structure of a stable fragment from BPAG1, residues 226-448, defined by limited proteolysis of the whole Plakin domain. The structure, determined by single-wavelength anomalous diffraction phasing from a selenomethionine-substituted crystal at 3.0 A resolution, reveals a tandem pair of triple helical bundles closely related to spectrin repeats. Based on this structure and analysis of sequence conservation, we propose that the architecture of Plakin domains is defined by two pairs of spectrin repeats interrupted by a putative Src-Homology 3 (SH3) domain.
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the role of microtubule actin cross linking factor 1 macf1 in the wnt signaling pathway
Genes & Development, 2006Co-Authors: Hui-jye Chen, Conrad L. Leung, Raul Perezolle, Ronald K.h. LiemAbstract:Plakins (spectraPlakins) are linker proteins that connect the cytoskeletal network to membrane-associated junctional complexes and are important in maintaining tissue integrity. MACF1 (microtubule actin cross-linking factor 1), also called ACF7 (actin cross-linking factor 7), and BPAG1 (bullous pemphigoid antigen 1) belong to the Plakin family along with desmoPlakin, plectin, envoPlakin, periPlakin, and epiPlakin (Leung et al. 2002). The transcripts of MACF1 and BPAG1 are alternatively spliced resulting in different isoforms with different functional domains. MACF1a and BPAG1a have similar domain organization with an actin-binding domain, a Plakin domain that can bind junctional complexes, a rod domain with spectrin repeats, and a C terminus that can bind and stabilize microtubules (Leung et al. 1999; Karakesisoglou et al. 2000; Sun et al. 2001). The alternatively spliced isoforms, MACF1b and BPAG1b, contain variable numbers of Plakin (or plectin) repeats between the Plakin domain and spectrin repeats. MACF1b is involved in maintaining the structure of the Golgi complex (Lin et al. 2005). In epithelial cells, another alternatively spliced isoform of BPAG1 (BPAG1e) forms a link between hemidesmosomes and keratin intermediate filaments. BPAG1−/− mice have sensory neuron defects due to the absence of BPAG1a in the nervous system. Even though BPAG1 is ubiquitously expressed, degeneration is only observed in sensory neurons, suggesting that MACF1 might compensate for BPAG1 in other regions. MACF1 homologs have been identified in Drosophila (shortstop or shot) and Caenorhabditis elegans (Vab-10). Mutations of shot result in defects in epidermal integrity, epidermal muscle attachment, muscle-dependent tendon cell differentiation, anastomosis of the tracheal branches, axonal outgrowth and guidance, and dendritic morphogenesis (for review, see Roper and Brown 2003). In C. elegans, Vab-10 mutants display elongation and body morphology defects (Bosher et al. 2003). MACF1 is expressed ubiquitously in mouse embryos, with the highest levels in the nervous system followed by skeletal muscles and myocardia (Leung et al. 1999). In keratinocytes, MACF1 colocalizes with microtubules and actin at the cell periphery and relocates to sites of cell–cell contact upon stimulation (Karakesisoglou et al. 2000). MACF1-null endodermal cells fail to sustain polarization signals and to coordinate cell migration in response to wounding (Kodama et al. 2003). The Wnt family of secreted glycoproteins is a family of signaling molecules that regulate development at different stages, from patterning of the embryo, generation of tissues, and specification of cell fate, to regulation of cell movement, polarity, axon guidance, and synapse formation (Packard et al. 2003; Strutt 2003; Veeman et al. 2003; Nusse 2005). In the absence of Wnt, a complex of β-catenin, Axin, GSK3β, and APC (adenomatous polyposis coli) is present in the cytoplasm. GSK3β phosphorylates β-catenin, thereby targeting it for degradation. When Wnt binds to its receptor Frizzled and coreceptor LRP5/6, Axin is recruited to the cell membrane where it binds LRP5 and is subsequently subjected to degradation. As a result, GSK3β no longer phosphorylates β-catenin, and β-catenin goes to the nucleus, where it complexes with TCF/LEF proteins to activate the transcription of Wnt target genes. We have generated MACF1−/− mice and found that they died prenatally. MACF1−/− embryos lacked a primitive streak, node, and mesoderm. This MACF1−/− phenotype was similar to the phenotypes of Wnt-3−/− and LRP5/6 double-knockout mice. MACF1 interacted directly with Axin and was involved in the translocation of the Axin complex from the cytoplasm to the plasma membrane. The absence of MACF1 blocked Axin translocation and inhibited the downstream β-catenin/TCF transcriptional activation, indicating that MACF1 acted as a positive regulator in the Wnt signaling pathway.
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Plakins: a family of versatile cytolinker proteins.
Trends in cell biology, 2002Co-Authors: Conrad L. Leung, Kathleen J Green, Ronald K.h. LiemAbstract:By connecting cytoskeletal elements to each other and to junctional complexes, the Plakin family of cytolinkers plays a crucial role in orchestrating cellular development and maintaining tissue integrity. Plakins are built from combinations of interacting domains that bind to microfilaments, microtubules, intermediate filaments, cell-adhesion molecules and members of the armadillo family. Plakins are involved in both inherited and autoimmune diseases that affect the skin, neuronal tissue, and cardiac and skeletal muscle. Here, we describe the members of the Plakin family and their interaction partners, and give examples of the cellular defects that result from their dysfunction.
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The Plakin family
Journal of Cell Science, 2001Co-Authors: Conrad L. Leung, Ronald K.h. Liem, David A. D. Parry, Kathleen J GreenAbstract:The Plakins (also referred to as cytolinkers) are a family of large, modular proteins that link cytoskeletal networks to each other and to membrane-associated adhesive junctions, such as desmosomes and hemidesmosomes. Mutations in Plakin family genes lead to defects in tissue integrity and function
Martyn Chidgey - One of the best experts on this subject based on the ideXlab platform.
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molecular mechanism of intermediate filament recognition by Plakin proteins
Biochimica et Biophysica Acta, 2020Co-Authors: Fiyaz Mohammed, Michael Overduin, Catharine A Trieber, Martyn ChidgeyAbstract:The Plakin family of cytolinkers interacts with intermediate filaments (IFs) through Plakin repeat domain (PRD) and linker modules. Recent structure/function studies have established the molecular basis of envoPlakin-PRD and periPlakin-linker interactions with vimentin. Both Plakin modules share a broad basic groove which recognizes acidic rod elements on IFs, a mechanism that is applicable to other Plakin family members. This review postulates a universal IF engagement mechanism that illuminates the specific effects of pathogenic mutations associated with diseases including arrhythmogenic right ventricular cardiomyopathy, and reveals how diverse Plakin proteins offer tailored IF tethering to ensure stable, dynamic and regulated cellular structures.
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Mechanism of intermediate filament recognition by Plakin repeat domains revealed by envoPlakin targeting of vimentin
Nature Communications, 2016Co-Authors: Claudia Fogl, Michael Overduin, Fiyaz Mohammed, Caezar Al-jassar, Mark Jeeves, Timothy J. Knowles, Penelope Rodriguez-zamora, Scott A. White, Elena Odintsova, Martyn ChidgeyAbstract:Plakin proteins form critical connections between cell junctions and the cytoskeleton; their disruption within epithelial and cardiac muscle cells cause skin-blistering diseases and cardiomyopathies. EnvoPlakin has a single Plakin repeat domain (PRD) which recognizes intermediate filaments through an unresolved mechanism. Herein we report the crystal structure of envoPlakin’s complete PRD fold, revealing binding determinants within its electropositive binding groove. Four of its five internal repeats recognize negatively charged patches within vimentin via five basic determinants that are identified by nuclear magnetic resonance spectroscopy. Mutations of the Lys1901 or Arg1914 binding determinants delocalize heterodimeric envoPlakin from intracellular vimentin and keratin filaments in cultured cells. Recognition of vimentin is abolished when its residues Asp112 or Asp119 are mutated. The latter slot intermediate filament rods into basic PRD domain grooves through electrosteric complementarity in a widely applicable mechanism. Together this reveals how Plakin family members form dynamic linkages with cytoskeletal frameworks. Plakin proteins link cell junctions to cytoskeletal frameworks, and their disruption within epithelial and cardiac muscle cells cause skin blistering diseases and cardiomyopathies. Here the authors use structural biology approaches to reveal the mechanism that allows Plakins to recognize intermediate filaments within the cytoskeleton.
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Mechanism of intermediate filament recognition by Plakin repeat domains revealed by envoPlakin targeting of vimentin.
Nature communications, 2016Co-Authors: Claudia Fogl, Michael Overduin, Fiyaz Mohammed, Caezar Al-jassar, Mark Jeeves, Timothy J. Knowles, Penelope Rodriguez-zamora, Scott A. White, Elena Odintsova, Martyn ChidgeyAbstract:Plakin proteins form critical connections between cell junctions and the cytoskeleton; their disruption within epithelial and cardiac muscle cells cause skin-blistering diseases and cardiomyopathies. EnvoPlakin has a single Plakin repeat domain (PRD) which recognizes intermediate filaments through an unresolved mechanism. Herein we report the crystal structure of envoPlakin's complete PRD fold, revealing binding determinants within its electropositive binding groove. Four of its five internal repeats recognize negatively charged patches within vimentin via five basic determinants that are identified by nuclear magnetic resonance spectroscopy. Mutations of the Lys1901 or Arg1914 binding determinants delocalize heterodimeric envoPlakin from intracellular vimentin and keratin filaments in cultured cells. Recognition of vimentin is abolished when its residues Asp112 or Asp119 are mutated. The latter slot intermediate filament rods into basic PRD domain grooves through electrosteric complementarity in a widely applicable mechanism. Together this reveals how Plakin family members form dynamic linkages with cytoskeletal frameworks.
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Sequence-specific 1H, 13C and 15N backbone resonance assignments of the Plakin repeat domain of human envoPlakin
Biomolecular NMR assignments, 2015Co-Authors: Mark Jeeves, Martyn Chidgey, Claudia Fogl, Caezar Al-jassar, Michael OverduinAbstract:The Plakin repeat domain is a distinctive hallmark of the Plakin superfamily of proteins, which are found within all epithelial tissues. Plakin repeat domains mediate the interactions of these proteins with the cell cytoskeleton and are critical for the maintenance of tissue integrity. Despite their biological importance, no solution state resonance assignments are available for any homologue. Here we report the essentially complete 1H, 13C and 15N backbone chemical shift assignments of the singular 22 kDa Plakin repeat domain of human envoPlakin, providing the means to investigate its interactions with ligands including intermediate filaments.
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hinged Plakin domains provide specialized degrees of articulation in envoPlakin periPlakin and desmoPlakin
PLOS ONE, 2013Co-Authors: Caezar Aljassar, Martyn Chidgey, Pau Bernadό, Michael OverduinAbstract:: EnvoPlakin, periPlakin and desmoPlakin are cytoskeletal proteins that provide structural integrity within the skin and heart by resisting shear forces. Here we reveal the nature of unique hinges within their Plakin domains that provides divergent degrees of flexibility between rigid long and short arms composed of spectrin repeats. The range of mobility of the two arms about the hinge is revealed by applying the ensemble optimization method to small-angle X-ray scattering data. EnvoPlakin and periPlakin adopt 'L' shaped conformations exhibiting a 'helicopter propeller'-like mobility about the hinge. By contrast desmoPlakin exhibits essentially unrestricted mobility by 'jack-knifing' about the hinge. Thus the diversity of molecular jointing that can occur about Plakin hinges includes 'L' shaped bends, 'U' turns and fully extended 'I' orientations between rigid blocks of spectrin repeats. This establishes specialised hinges in Plakin domains as a key source of flexibility that may allow sweeping of cellular spaces during assembly of cellular structures and could impart adaptability, so preventing irreversible damage to desmosomes and the cell cytoskeleton upon exposure to mechanical stress.
Hala Zahreddine - One of the best experts on this subject based on the ideXlab platform.
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crt 1 calreticulin and the e3 ligase eel 1 huwe1 control hemidesmosome maturation in c elegans development
Current Biology, 2010Co-Authors: Hala Zahreddine, Marie Diogon, Yasuko Nagamatsu, Huimin Zhang, Michel LabouesseAbstract:Summary Hemidesmosomes connect the extracellular matrix (ECM) to intermediate filaments through ECM receptors and Plakins (plectin and BPAG1e). They affect tissue integrity, wound healing, and carcinoma invasion [1]. Although biochemical and time-lapse studies indicate that α6β4-integrin (ECM receptor) and plectin play a central role in modulating hemidesmosome disassembly [2–5], the mechanisms controlling hemidesmosome biogenesis in vivo remain poorly understood. The nematode C. elegans provides a powerful genetic model to address this issue. We performed a genome-wide RNA interference screen in C. elegans , searching for genes that decrease the viability of a weak VAB-10A/Plakin mutant. We identified 14 genes that have human homologs with predicted roles in different cellular processes. We further characterized two genes encoding the chaperone CRT-1/calreticulin and the HECT domain E3 ubiquitin ligase EEL-1/HUWE1. CRT-1 controls by as little as 2-fold the abundance of UNC-52/perlecan, an essential hemidesmosome ECM ligand. Likewise, EEL-1 fine tunes by 2-fold the abundance of myotactin, the putative hemidesmosome ECM receptor. CRT-1 and EEL-1 activities, and by extension other genes identified in our screen, are essential during embryonic development to enable hemidesmosomes exposed to mechanical tension to mature into a tension-resistant form. Our findings should help understand how hemidesmosome dynamics are regulated in vertebrate systems.
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CRT-1/calreticulin and the E3 ligase EEL-1/HUWE1 control hemidesmosome maturation in C. elegans development.
Current Biology - CB, 2010Co-Authors: Hala Zahreddine, Marie Diogon, Yasuko Nagamatsu, Huimin Zhang, Michel LabouesseAbstract:Hemidesmosomes connect the extracellular matrix (ECM) to intermediate filaments through ECM receptors and Plakins (plectin and BPAG1e). They affect tissue integrity, wound healing, and carcinoma invasion. Although biochemical and time-lapse studies indicate that alpha6beta4-integrin (ECM receptor) and plectin play a central role in modulating hemidesmosome disassembly, the mechanisms controlling hemidesmosome biogenesis in vivo remain poorly understood. The nematode C. elegans provides a powerful genetic model to address this issue. We performed a genome-wide RNA interference screen in C. elegans, searching for genes that decrease the viability of a weak VAB-10A/Plakin mutant. We identified 14 genes that have human homologs with predicted roles in different cellular processes. We further characterized two genes encoding the chaperone CRT-1/calreticulin and the HECT domain E3 ubiquitin ligase EEL-1/HUWE1. CRT-1 controls by as little as 2-fold the abundance of UNC-52/perlecan, an essential hemidesmosome ECM ligand. Likewise, EEL-1 fine tunes by 2-fold the abundance of myotactin, the putative hemidesmosome ECM receptor. CRT-1 and EEL-1 activities, and by extension other genes identified in our screen, are essential during embryonic development to enable hemidesmosomes exposed to mechanical tension to mature into a tension-resistant form. Our findings should help understand how hemidesmosome dynamics are regulated in vertebrate systems.
Jose M De Pereda - One of the best experts on this subject based on the ideXlab platform.
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Architecture of hemidesmosomes
'Sociedad Espanola de Bioquimica y Biologia Molecular (SEBBM)', 2018Co-Authors: Manso, José A., Carabias Arturo, Sonnenberg Arnoud, Gómez-hernández María, García-rubio Inés, Jose M De PeredaAbstract:Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016.Hemidesmosomes (HDs) are junctional complexes that mediate stable attachment of epithelial cells to the basal lamina, linking the extracellular matrix to the cytokeratins. In stratified epithelia HDs are composed of integrin α6β4, the bullous pemphigoid antigen 2 (BPAG2), tetraspanin CD151, and two proteins of the Plakin family: plectin and BPAG1e. The integrin α6β4 is a receptor for laminins and a central hub of the HD protein network. The intracellular interactions of α6β4 are mediated by the cytodomain of the β4 subunit. Plectin and BPAG1e bind to β4 via their N-terminal regions and to cytokeratins via their C-terminal domains. Defects in HD-proteins cause several types of the blistering disease epidermolysis bullosa. Our long-term goal is to understand the organization and regulation of HDs. Previously, we had characterized the structure of the plectin-integrin β4 complex. Now we have elucidated the structural basis of the BPAG1e-β4 interaction. Binding occurs between an N-terminal segment of BPAG1e and the third and fourth fibronectin type III domains (FnIII-3,4) of β4. Ser residues in BPAG1e are involved in the binding interface, suggesting that the interaction may be regulated by phosphorylation. Complementing the analysis of hetero-interactions, we have elucidated the structure of dimeric Plakins. Similarly to most Plakins, plectin has an N-terminal “Plakin domain” formed by spectrin repeats that adopt an elongated structure, which is followed by a central coiled-coil rod domain that mediates dimerization. The Plakin domains are arranged in parallel in the homo-dimer. Thus, the distance between the β4 binding-sites is restricted, suggesting that the avidity-mediated stabilization of the interaction might be linked to the clustering of α6β4 in the membrane.Peer Reviewe
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Structure of a dimeric fragment of the Plakin domain of plectin by hybrid methods
2018Co-Authors: Carabias Arturo, Carballido Vázquez, Ana M., Manso, José A., García-rubio Inés, Jose M De PeredaAbstract:Resumen del póster presentado al 5th International Iberian Biophysics Congress, celebrado en Porto (Portugal) del 15 al 17 de junio de 2016.Plectin is a member of the Plakin family of high molecular weight cytolinkers. Plectin has a tripartite structure with N- and C-terminal regions separated by a central rod domain. The N-terminal region contains an actin binding domain (ABD) and a Plakin domain. The ABD binds to integrin α6ß4 in hemidesmosomes and to the nuclear envelope protein nesprin-3. The Plakin domain is formed by nine spectrin repeats (SR1-SR9) and an SH3 domain. Each SR consists of three α-helices (A-B-C) arranged in a left-handed bundle. Helix C of a SR is fussed to helix A of the downstream repeat; hence, the Plakin domain has an elongated shape. Downstream of SR9, the rod domain forms a parallel coiled-coil that mediates dimerization. Here, we have combined hybrid methods to elucidate the structure of a dimeric fragment of plectin that includes the SR7-SR9, and the initial region of the rod domain. The crystal structure revealed contacts along the Plakin domain between two protomers. Analysis by small angle X-ray scattering (SAXS) supports that the closed arrangement of the dimer also occurs in solution. This was further confirmed by measuring eight inter-monomer distances combining site directed spin labelling and double electron-electron resonance spectroscopy (EPR-DEER). Collectively, our data suggest that the Plakin domain contributes to the stabilization of the plectin dimer. Moreover, the rigid structure of the SR3-SR9 constraints the maximum spacing between the binding-sites for integrin ß4 and nesprin-3 in the dimer, suggesting that avidity-driven recruitment of plectin might depend on the density of the interaction partners.Peer Reviewe
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The structure of the Plakin domain of plectin reveals an extended rod-like shape
'American Society for Biochemistry & Molecular Biology (ASBMB)', 2018Co-Authors: Ortega Esther, Manso, José A., Buey, Ruben M., Carballido Vázquez, Ana M., Carabias Arturo, Sonnenberg Arnoud, Jose M De PeredaAbstract:Plakins are large multi-domain proteins that interconnect cytoskeletal structures. Plectin is a prototypical Plakin that tethers intermediate filaments to membrane-associated complexes. Most Plakins contain a Plakin domain formed by up to nine spectrin repeats (SR1-SR9) and an SH3 domain. The Plakin domains of plectin and other Plakins harbor binding sites for junctional proteins. We have combined x-ray crystallography with small angle x-ray scattering (SAXS) to elucidate the structure of the Plakin domain of plectin, extending our previous analysis of the SR1 to SR5 region. Two crystal structures of the SR5-SR6 region allowed us to characterize its uniquely wide inter-repeat conformational variability. We also report the crystal structures of the SR7-SR8 region, refined to 1.8 Å, and the SR7-SR9 at lower resolution. The SR7-SR9 region, which is conserved in all other Plakin domains, forms a rigid segment stabilized by uniquely extensive inter-repeat contacts mediated by unusually long helices in SR8 and SR9. Using SAXS we show that in solution the SR3-SR6 and SR7-SR9 regions are rod-like segments and that SR3-SR9 of plectin has an extended shape with a small central kink. Other Plakins, such as bullous pemphigoid antigen 1 and microtubule and actin cross-linking factor 1, are likely to have similar extended Plakin domains. In contrast, desmoPlakin has a two-segment structure with a central flexible hinge. The continuous versus segmented structures of the Plakin domains of plectin and desmoPlakin give insight into how different Plakins might respond to tension and transmit mechanical signals.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the European Regional Development Fund, Grants BFU2012-32847 and BFU2015-69499-P (to J. M. dP), as well as by the Netherlands Science Foundation and the Dutch Cancer Society (to A. S.). This work also was supported by the European Community's Seventh Framework Programme (FP7/2007–2013) under BioStruct-X Grant Agreement 283570.Peer Reviewe
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Structure of the Plakin domain of plectin: implications for the mechanical properties of Plakins
2018Co-Authors: Ortega Esther, Manso, José A., Buey, Ruben M., Carballido Vázquez, Ana M., Carabias Arturo, Sonnenberg Arnoud, Jose M De PeredaAbstract:Resumen del póster presentado a la CNIC Conference: Mechanical forces in physiology and disease, celebrada en Madrid (España) del 4 al 5 de noviembre de 2016.Plakins are large multi-domain proteins that interconnect cytoskeletal structures. Plectin is a prototypical Plakin that tethers intermediate filaments to membrane-associated complexes. For example in epithelia plectin links the cytokeratins to the integrin α6ß4 in the hemidesmosomes. The N-terminal region of plectin contains an actin binding domain that mediates the binding to α6ß4, and a region named the Plakin domain that is conserved in most Plakins. The Plakin domain of plectin is formed by nine spectrin repeats (SR1 to SR9) and a non-canonical SH3 domain. We have combined X-ray crystallography with small angle X-ray scattering (SAXS) to elucidate the global structure of the Plakin domain of plectin, extending our previous analysis of the SR1 to SR5 region. Two crystal structures of the SR5-SR6 segment allowed us to characterize its uniquely wide inter-repeat conformational variability. We also report the crystal structures of the SR7-SR8 region, refined to 1.8 Å, and the SR7-SR9 at lower resolution. The SR7-SR9 region, which is conserved in all other Plakin domains, forms a rigid segment stabilized by uniquely extensive inter-repeat contacts mediated by unusually long helices in SR8 and SR9. Using SAXS we show that in solution the SR3-SR9 of plectin has an extended shape with a small central kink. Other Plakins, such as bullous pemphigoid antigen 1 (BPAG1) and microtubule and actin crosslinking factor 1 (MACF1), are likely to have similar extended Plakin domains. The array of SRs in the Plakin domain is very similar to those present in spectrins. Such arrays of SRs form deformable structures that can be bent; SRs can also unfold individually at low pulling forces. The continuous and extended rod-like structure of plectin suggests that the Plakin domain may work as a molecular shock absorbent that dissipates elastic energy when cells are subjected to external forces, and might contribute to the mechanical stability and resilience of tissues that are subjected to mechanical stressPeer Reviewe
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Purification and structural analysis of plectin and BPAG1e
'Elsevier BV', 2018Co-Authors: Manso, José A., Ortega Esther, Buey, Ruben M., Carballido Vázquez, Ana M., Carabias Arturo, Gómez-hernández María, García-rubio Inés, Alonso-garcía Noelia, Jose M De PeredaAbstract:Plectin and BPAG1e belong to the Plakin family of high-molecular-weight proteins that interconnect the cytoskeletal systems and anchor them to junctional complexes. Plectin and BPAG1e are prototypical Plakins with a similar tripartite modular structure. The N- and C-terminal regions are built of multiple discrete structural domains, while the central rod domain mediates dimerization by coiled-coil interactions. Owing to the mosaic organization of Plakins, the structure of their constituent individual domains or small multi-domain segments can be analyzed isolated. Yet, understanding the integrated function of large regions, oligomers, and heterocomplexes of Plakins is difficult due to the large and segmented structure. Here, we describe methods for the production of plectin and BPAG1e samples suitable for structural and biophysical analysis. In addition, we discuss the combination of hybrid methods that yield information at several resolution levels to study the complex, multi-domain, and flexible structure of Plakins.This work was supported by the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (grant BFU2012-32847 to J. M. d. P.).Peer Reviewe