The Experts below are selected from a list of 321 Experts worldwide ranked by ideXlab platform

Vann Bennett - One of the best experts on this subject based on the ideXlab platform.

  • Structural basis of diverse membrane target recognitions by Ankyrins
    eLife, 2014
    Co-Authors: Chao Wang, Vann Bennett, Zhiyi Wei, Keyu Chen, Mingjie Zhang
    Abstract:

    Proteins are made up of smaller building blocks called amino acids that are linked to form long chains that then fold into specific shapes. Each protein gets its unique identity from the number and order of the amino acids that it contains, but different proteins can contain similar arrangements of amino acids. These similar sequences, known as motifs, are usually short and typically mark the sites within proteins that bind to other molecules or proteins. A single protein can contain many motifs, including multiple repeats of the same motif. One common motif is called the Ankyrin (or ANK) repeat, which is found in 100s of proteins in different species, including bacteria and humans. Ankyrin proteins perform a range of important functions, such as connecting proteins in the cell surface membrane to a scaffold-like structure underneath the membrane. Proteins containing Ankyrin repeats are known to interact with a diverse range of other proteins (or targets) that are different in size and shape. The 24 repeats found in human Ankyrin proteins appear to have essentially remained unchanged for the last 500 million years. As such, it remains unclear how the conserved Ankyrin repeats can bind to such a wide variety of protein targets. Now, Wang, Wei et al. have uncovered the three-dimensional structure of Ankyrin repeats from a human Ankyrin protein while it was bound either to a regulatory fragment from another Ankyrin protein or to a region of a target protein (which transports sodium ions in and out of cells). The Ankyrin repeats were shown to form an extended ‘left-handed helix’: a structure that has also been seen in other proteins with different repeating motifs. Wang, Wei et al. found that the Ankyrin protein fragment bound to the inner surface of the part of the helix formed by the first 14 Ankyrin repeats. The target protein region also bound to the helix's inner surface. Wang, Wei et al. show that this surface contains many binding sites that can be used, in different combinations, to allow Ankyrins to interact with diverse proteins. Other proteins with long sequences of repeats are widespread in nature, but uncovering the structures of these proteins is technically challenging. Wang, Wei et al.'s findings might reveal new insights into the functions of many of such proteins in a wide range of living species. Furthermore, the new structures could help explain why specific mutations in the genes that encode Ankyrins (or their binding targets) can cause various diseases in humans—including heart diseases and psychiatric disorders.

  • GLIAL AnkyrinS FACILITATE PARANODAL AXOGLIAL JUNCTION ASSEMBLY
    Nature neuroscience, 2014
    Co-Authors: Kae-jiun Chang, Vann Bennett, Peter J Mohler, Daniel R. Zollinger, Keiichiro Susuki, Diane L. Sherman, Michael A. Makara, Peter J. Brophy, Edward C. Cooper, Matthew N. Rasband
    Abstract:

    In this study, the authors show that the scaffolding proteins Ankyrin B and Ankyrin G are expressed by Schwann cells and oligodendrocytes, respectively, and are enriched on the glial membrane at paranodal junctions where they interact with neurofascin 155. In addition, they find that Ankyrins in oligodendrocytes have key roles in rapid and efficient paranode formation in the CNS.

  • A single divergent exon inhibits Ankyrin-B association with the plasma membrane.
    The Journal of biological chemistry, 2013
    Co-Authors: Wei Chou Tseng, Vann Bennett
    Abstract:

    Abstract Vertebrate Ankyrin-B and Ankyrin-G exhibit divergent subcellular localization and function despite their high sequence and structural similarity and common origin from a single ancestral gene at the onset of chordate evolution. Previous studies of Ankyrin family diversity have focused on the C-terminal regulatory domain. Here, we identify an Ankyrin-B-specific linker peptide connecting the Ankyrin repeat domain to the ZU52-UPA module that inhibits binding of Ankyrin-B to membrane protein partners E-cadherin and neurofascin 186 and prevents association of Ankyrin-B with epithelial lateral membranes as well as neuronal plasma membranes. The residues of the Ankyrin-B linker required for autoinhibition are encoded by a small exon that is highly divergent between Ankyrin family members but conserved in the Ankyrin-B lineage. We show that the Ankyrin-B linker suppresses activity of the ANK repeat domain through an intramolecular interaction, likely with a groove on the surface of the ANK repeat solenoid, thereby regulating the affinities between Ankyrin-B and its binding partners. These results provide a simple evolutionary explanation for how Ankyrin-B and Ankyrin-G have acquired striking differences in their plasma membrane association while maintaining overall high levels of sequence similarity.

  • Spectrin- and Ankyrin-based membrane domains and the evolution of vertebrates.
    Current topics in membranes, 2013
    Co-Authors: Vann Bennett, Damaris N. Lorenzo
    Abstract:

    Abstract Spectrin and Ankyrin are membrane skeletal proteins that contribute to mechanical support of plasma membranes and micron-scale organization of diverse membrane-spanning proteins. This chapter provides a plausible scenario for the evolution of Ankyrin- and spectrin-based membrane domains with a focus on vertebrates. The analysis integrates recent phylogenetic information with functional analyses of spectrin and Ankyrin in erythrocytes, axon initial segments and nodes of Ranvier in neurons, T-tubules and intercalated disks of cardiomyocytes, lateral membrane domains of epithelial cells, and costameres of striated muscle. A core spectrin–Ankyrin mechanism for coordinating membrane-spanning proteins and mechanically stabilizing membrane bilayers was expanded in vertebrates by gene duplication events, insertion of giant alternately spliced exons of axonal Ankyrins, and a versatile peptide-binding fold of ANK repeats that facilitated acquisition of new protein partners. Cell adhesion molecules (CAM), including dystroglycan, L1 CAM family members, and cadherins, are the earliest examples of membrane-spanning proteins with Ankyrin-binding motifs and were all present in urochordates. In contrast, ion channels have continued to evolve Ankyrin-binding sites in vertebrates. These considerations suggest a model where proto-domains formed through interaction of Ankyrin and spectrin with CAMs. These proto-domains then became populated with ion channels that developed Ankyrin-binding activity with selective pressure provided by optimization of physiological function. The best example is the axon initial segment where Ankyrin-binding activity evolved sequentially and independently first in L1 CAMs, then in voltage-gated sodium channels, and finally in KCNQ2/3 channels, with the selective advantage of fast and precisely regulated signaling.

  • Ankyrin-B structurally defines terminal microdomains of peripheral somatosensory axons
    Brain structure & function, 2012
    Co-Authors: Maren Engelhardt, Vann Bennett, Silke Vorwald, Jürgen-markus Sobotzik, Christian Schultz
    Abstract:

    Axons are subdivided into functionally organized microdomains, which are required for generation and propagation of action potentials (APs). In the central nervous system (CNS), APs are generated near the soma in the axon initial segment (AIS) and propagated by nodes of Ranvier (noR). The crucial role of the membrane adapter proteins Ankyrin-B and Ankyrin-G as organizers of AIS and noR is now well established. By comparison, little is known on the localization and function of these proteins in sensory axon terminals of the peripheral nervous systems (PNS). Here, we tested the hypothesis that somatosensory PNS terminals are organized by distinct members of the Ankyrin protein family. We discovered a specific distribution of Ankyrin-B in somatosensory axon terminals of skin and muscle. Specifically, Ankyrin-B was localized along the membrane of axons innervating Meissner corpuscles, Pacinian corpuscles and hair follicle receptors. Likewise, proprioceptive terminals of muscle spindles exhibited prominent Ankyrin-B expression. Furthermore, Ankyrin-B expression extended into nociceptive and thermoceptive intraepidermal nerve fibers. Interestingly, all studied somatosensory terminals were largely devoid of Ankyrin-G, indicating that this scaffolding protein does not contribute to organization of mechanoelectric transduction zones in peripheral somatosensory neurons. Instead, we propose that Ankyrin-B serves as a major membrane organizer in mechanoreceptive and nociceptive terminals of the PNS.

Peter J Mohler - One of the best experts on this subject based on the ideXlab platform.

  • Ankyrins and Spectrins in Cardiovascular Biology and Disease.
    Frontiers in physiology, 2017
    Co-Authors: Mona El Refaey, Peter J Mohler
    Abstract:

    Ankyrins are adaptor proteins critical for the expression and targeting of cardiac membrane proteins, signaling molecules, and cytoskeletal elements. Findings in humans and animal models have highlighted the in vivo roles for Ankyrins in normal physiology and in cardiovascular disease, most notably in cardiac arrhythmia. For example, human ANK2 loss-of-function variants are associated with a complex array of electrical and structural phenotypes now termed "Ankyrin-B syndrome," whereas alterations in the Ankyrin-G pathway for Nav channel targeting are associated with human Brugada syndrome. Further, both Ankyrin-G and -B are now linked with acquired forms of cardiovascular disease including myocardial infarction and atrial fibrillation. Spectrins are Ankyrin-associated proteins and recent studies support the critical role of Ankyrin-spectrin interactions in normal cardiac physiology as well as regulation of key ion channel and signaling complexes. This review will highlight the roles of Ankyrins and spectrins in cardiovascular physiology as well as illustrate the link between the dysfunction in Ankyrin- and spectrin-based pathways and disease.

  • The evolving role of Ankyrin-B in cardiovascular disease
    Heart rhythm, 2017
    Co-Authors: Sara N. Koenig, Peter J Mohler
    Abstract:

    Over the past decade, Ankyrin-B has been identified as a prominent player in cardiac physiology. Ankyrin-B has a multitude of functions, with roles in expression, localization, and regulation of proteins critical for cardiac excitability, cytoskeletal integrity, and signaling. Furthermore, human ANK2 variants that result in Ankyrin-B loss of function are associated with "Ankyrin-B syndrome," a complex cardiac phenotype that may include bradycardia and heart rate variability, conduction block, atrial fibrillation, QT interval prolongation, and potentially fatal catecholaminergic polymorphic ventricular tachycardia. However, our understanding of the molecular mechanisms underlying Ankyrin-B function at baseline and in disease is still not fully developed owing to the complexity of Ankyrin-B gene regulation, number of Ankyrin-B–associated molecules, multiple roles of Ankyrin-B in the heart and other organs that modulate cardiac function, and a host of unexpected clinical phenotypes. In this review, we summarize known roles of Ankyrin-B in the heart and the impact of Ankyrin-B dysfunction in animal models and in human disease as well as highlight important new findings illustrating the complexity of Ankyrin-B signaling.

  • GLIAL AnkyrinS FACILITATE PARANODAL AXOGLIAL JUNCTION ASSEMBLY
    Nature neuroscience, 2014
    Co-Authors: Kae-jiun Chang, Vann Bennett, Peter J Mohler, Daniel R. Zollinger, Keiichiro Susuki, Diane L. Sherman, Michael A. Makara, Peter J. Brophy, Edward C. Cooper, Matthew N. Rasband
    Abstract:

    In this study, the authors show that the scaffolding proteins Ankyrin B and Ankyrin G are expressed by Schwann cells and oligodendrocytes, respectively, and are enriched on the glial membrane at paranodal junctions where they interact with neurofascin 155. In addition, they find that Ankyrins in oligodendrocytes have key roles in rapid and efficient paranode formation in the CNS.

  • Ankyrin g participates in ina remodeling in myocytes from the border zones of infarcted canine heart
    PLOS ONE, 2013
    Co-Authors: Wen Dun, Peter J Mohler, John S. Lowe, Thomas J. Hund, Patrick J Wright, Penelope A Boyden
    Abstract:

    Cardiac Na channel remodeling provides a critical substrate for generation of reentrant arrhythmias in border zones of the infarcted canine heart. Recent studies show that Nav1.5 assembly and function are linked to Ankyrin-G, gap, and mechanical junction proteins. In this study our objective is to expound the status of the cardiac Na channel, its interacting protein AnkyrinG and the mechanical and gap junction proteins at two different times post infarction when arrhythmias are known to occur; that is, 48 hr and 5 day post coronary occlusion. Previous studies have shown the origins of arrhythmic events come from the subendocardial Purkinje and epicardial border zone. Our Purkinje cell (Pcell) voltage clamp study shows that INa and its kinetic parameters do not differ between Pcells from the subendocardium of the 48hr infarcted heart (IZPCs) and control non-infarcted Pcells (NZPCs). Immunostaining studies revealed that disturbances of Nav1.5 protein location with Ankyrin-G are modest in 48 hr IZPCs. Therefore, Na current remodeling does not contribute to the abnormal conduction in the subendocardial border zone 48 hr post myocardial infarction as previously defined. In addition, immunohistochemical data show that Cx40/Cx43 co-localize at the intercalated disc (IDs) of control NZPCs but separate in IZPCs. At the same time, Purkinje cell desmoplakin and desmoglein2 immunostaining become diffuse while plakophilin2 and plakoglobin increase in abundance at IDs. In the epicardial border zone 5 days post myocardial infarction, immunoblot and immunocytochemical analyses showed that Ankyrin-G protein expression is increased and re-localized to submembrane cell regions at a time when Nav1.5 function is decreased. Thus, Nav1.5 and Ankyrin-G remodeling occur later after myocardial infarction compared to that of gap and mechanical junctional proteins. Gap and mechanical junctional proteins remodel in IZPCs early, perhaps to help maintain Nav1.5 subcellular location position and preserve its function soon after myocardial infarction.

  • Coordinating electrical activity of the heart: Ankyrin polypeptides in human cardiac disease.
    Expert opinion on therapeutic targets, 2011
    Co-Authors: Jerry Curran, Peter J Mohler
    Abstract:

    Introduction: Over the past ten years, Ankyrin polypeptides have emerged as players in cardiac excitation–contraction coupling. Once thought to solely play a structural role, loss-of-function variants of genes encoding Ankyrin polypeptides have highlighted how this protein mediates subcellular localization of various electrical components of the excitation–contraction coupling machinery. Evidence has revealed how disruption of this localization is the primary cause of various cardiomyopathies, ranging from long-QT syndrome 4, to sinus node disease, to more common forms of arrhythmias. Areas covered: The roles of Ankyrin polypeptides in excitation–contraction coupling in the heart and the development of Ankyrin-specific cardiomyopathies. How Ankyrin polypeptides may be involved in structural and electrical remodeling of the heart, post-myocardial infarct. How Ankyrin interactions with membrane-bound ion channels may regulate these channels' response to stimuli. New data, which offers the potential for uniq...

Jonathan Q. Davis - One of the best experts on this subject based on the ideXlab platform.

  • An Ankyrin-based mechanism for functional organization of dystrophin and dystroglycan.
    Cell, 2008
    Co-Authors: Gai Ayalon, Jonathan Q. Davis, Paula Scotland, Vann Bennett
    Abstract:

    Summary β-dystroglycan (DG) and the dystrophin-glycoprotein complex (DGC) are localized at costameres and neuromuscular junctions in the sarcolemma of skeletal muscle. We present evidence for an Ankyrin-based mechanism for sarcolemmal localization of dystrophin and β-DG. Dystrophin binds Ankyrin-B and Ankyrin-G, while β-DG binds Ankyrin-G. Dystrophin and β-DG require Ankyrin-G for retention at costameres but not delivery to the sarcolemma. Dystrophin and β-DG remain intracellular in Ankyrin-B-depleted muscle, where β-DG accumulates in a juxta-TGN compartment. The neuromuscular junction requires Ankyrin-B for localization of dystrophin/utrophin and β-DG and for maintenance of its postnatal morphology. A Becker muscular dystrophy mutation reduces Ankyrin binding and impairs sarcolemmal localization of dystrophin-Dp71. Ankyrin-B also binds to dynactin-4, a dynactin subunit. Dynactin-4 and a subset of microtubules disappear from sarcolemmal sites in Ankyrin-B-depleted muscle. Ankyrin-B thus is an adaptor required for sarcolemmal localization of dystrophin, as well as dynactin-4.

  • Ankyrin g is a molecular partner of e cadherin in epithelial cells and early embryos
    Journal of Biological Chemistry, 2007
    Co-Authors: Jonathan Q. Davis, Lydia Davis, Krishnakumar Kizhatil, Jan Hoffman, Brigid L M Hogan, Vann Bennett
    Abstract:

    E-cadherin is a ubiquitous component of lateral membranes in epithelial tissues and is required to form the first lateral membrane domains in development. Here, we identify Ankyrin-G as a molecular partner of E-cadherin and demonstrate that Ankyrin-G and β-2-spectrin are required for accumulation of E-cadherin at the lateral membrane in both epithelial cells and early embryos. Ankyrin-G binds to the cytoplasmic domain of E-cadherin at a conserved site distinct from that of β-catenin. Ankyrin-G also recruits β-2-spectrin to E-cadherin-β-catenin complexes, thus providing a direct connection between E-cadherin and the spectrin/actin skeleton. In addition to restricting the membrane mobility of E-cadherin, Ankyrin-G and β-2-spectrin also are required for exit of E-cadherin from the trans-Golgi network in a microtubule-dependent pathway. Ankyrin-G and β-2-spectrin co-localize with E-cadherin in preimplantation mouse embryos. Moreover, knockdown of either Ankyrin-G or β-2-spectrin in one cell of a two-cell embryo blocks accumulation of E-cadherin at sites of cell-cell contact. E-cadherin thus requires both Ankyrin-G and β-2-spectrin for its cellular localization in early embryos as well as cultured epithelial cells. We have recently reported that Ankyrin-G and β-2-spectrin collaborate in biogenesis of the lateral membrane(Kizhatil, K., Yoon, W., Mohler, P. J., Davis, L. H., Hoffman, J. A., and Bennett, V. (2007)J. Biol. Chem. 282 2029-2037. Together with the current findings, these data suggest a Ankyrin/spectrin-based mechanism for coordinating membrane assembly with extracellular interactions of E-cadherin at sites of cell-cell contact.

  • Ankyrin b coordinates the na k atpase na ca exchanger and insp3 receptor in a cardiac t tubule sr microdomain
    PLOS Biology, 2005
    Co-Authors: Peter J Mohler, Jonathan Q. Davis, Vann Bennett
    Abstract:

    We report identification of an Ankyrin-B-based macromolecular complex of Na/K ATPase (alpha 1 and alpha 2 isoforms), Na/Ca exchanger 1, and InsP3 receptor that is localized in cardiomyocyte T-tubules in discrete microdomains distinct from classic dihydropyridine receptor/ryanodine receptor “dyads.” E1425G mutation of Ankyrin-B, which causes human cardiac arrhythmia, also blocks binding of Ankyrin-B to all three components of the complex. The Ankyrin-B complex is markedly reduced in adult Ankyrin-B+/− cardiomyocytes, which may explain elevated [Ca2+]i transients in these cells. Thus, loss of the Ankyrin-B complex provides a molecular basis for cardiac arrhythmia in humans and mice. T-tubule-associated Ankyrin-B, Na/Ca exchanger, and Na/K ATPase are not present in skeletal muscle, where Ankyrin-B is expressed at 10-fold lower levels than in heart. Ankyrin-B also is not abundantly expressed in smooth muscle. We propose that the Ankyrin-B-based complex is a specialized adaptation of cardiomyocytes with a role for cytosolic Ca2+ modulation.

  • Isoform specificity among Ankyrins. An amphipathic alpha-helix in the divergent regulatory domain of Ankyrin-b interacts with the molecular co-chaperone Hdj1/Hsp40.
    The Journal of biological chemistry, 2004
    Co-Authors: Peter J Mohler, Jonathan Q. Davis, Janis A. Hoffman, Khadar Abdi, Chong-rak Kim, Sarah K. Jones, Lydia Davis, Karen F. Roberts, Vann Bennett
    Abstract:

    Abstract Ankyrins-R, -B, and -G are a family of membrane-associated adaptors required for localization of structurally diverse proteins to specialized membrane domains, including axon initial segments, cardiomyocyte T-tubules, and epithelial cell lateral membranes. Ankyrins are often co-expressed in the same cells and, although structurally similar, have non-overlapping functions. We previously determined that the regulatory domain of Ankyrin-B defines specificity between Ankyrins B and G in cardiomyocytes. Here, we identify key residues on the surface of an amphipathic α-helix unique to the regulatory domain of Ankyrin-B that are essential for the function of Ankyrin-B in cardiomyocytes. Using circular dichroism, we determined that a peptide representing the predicted helix folds as a helix in solution. Alanine-scanning mutagenesis revealed that residues 1773, 1777, 1780, 1784, and 1788 located in a patch on one surface the helix are critical for Ankyrin-B function in cardiomyocytes. In a parallel set of experiments we determined that the molecular co-chaperone human DnaJ homologue 1 (Hdj1)/Hsp40 interacts with the Ankyrin-B regulatory domain. Moreover, interaction of Hdj1/Hsp40 with the regulatory domain was mapped by random mutagenesis to same surface of the α-helix that is required for Ankyrin-B function. These results provide new insight into the molecular basis for specificity between Ankyrin-based pathways by defining a key α-helix structure in the divergent regulatory domain of Ankyrin-B as well as interaction of the helix with Hdj1/Hsp40, the first downstream target for Ankyrin-B-specific function.

  • Structural requirements for association of neurofascin with Ankyrin.
    The Journal of biological chemistry, 1998
    Co-Authors: Xu Zhang, Jonathan Q. Davis, Scott Carpenter, Vann Bennett
    Abstract:

    Abstract This paper presents the first structural analysis of the cytoplasmic domain of neurofascin, which is highly conserved among the L1CAM family of cell adhesion molecules, and describes sequence requirements for neurofascin-Ankyrin interactions in living cells. The cytoplasmic domain of neurofascin dimerizes in solution, has an asymmetric shape, and exhibits a reversible temperature-dependent β-structure. Residues Ser56–Tyr81 are necessary for Ankyrin binding but do not contribute to either dimerization or formation of structure. Transfected neurofascin recruits GFP-tagged 270-kDa AnkyrinG to the plasma membrane of human embryo kidney 293 cells. Deletion mutants demonstrate that the sequence Ser56–Tyr81 contains the major Ankyrin-recruiting activity of neurofascin. Mutations of the FIGQY tyrosine (Y81H/A/E) greatly impair neurofascin-Ankyrin interactions. Mutation of human L1 at the equivalent tyrosine (Y1229H) is responsible for certain cases of mental retardation (Van Camp, G., Fransen, E., Vits, L., Raes, G., and Willems, P. J. (1996) Hum. Mutat. 8, 391). Mutations F77A and E73Q greatly impair Ankyrin binding activity, whereas mutation D74N and a triple mutation of D57N/D58N/D62N result in less loss of Ankyrin binding activity. These results provide evidence for a highly specific interaction between Ankyrin and neurofascin and suggest that Ankyrin association with L1 is required for L1 function in humans.

Ekaterini Kordeli - One of the best experts on this subject based on the ideXlab platform.

  • Molecular Evolution of Ankyrin: Gain of Function in Vertebrates by Acquisition of an Obscurin/Titin-Binding-related Domain (OTBD).
    Molecular Biology and Evolution, 2006
    Co-Authors: Alexander Hopitzan, Anthony Baines, Ekaterini Kordeli
    Abstract:

    Ankyrins form a family of modular adaptor proteins that link between integral membrane proteins and the cytoskeleton. They evolved within the metazoa as an adaptation for organizing membrane microstructure and directing membrane traffic. Molecular cloning has identified one Caenorhabditis elegans (unc-44), two Drosophila (Dank1, Dank2) and three mammalian (Ank1, Ank2, Ank3) genes. We have previously identified a 76 amino acid alternatively spliced sequence that is present in muscle polypeptides encoded by the rat Ank3 gene. A closely related sequence in a muscle Ank1 product binds the cytoskeletal muscle proteins obscurin and titin. This Obscurin/Titin-Binding-related Domain (OTBD) contains repeated modules of 18 amino acids: three are encoded by Ank1 and Ank2, two by Ank3; this pattern is conserved throughout vertebrate Ankyrin genes. The Caenorhabditis elegans Ankyrin, UNC-44, contains one 18 amino acid module, as does the Ankyrin gene in the urochordate Ciona intestinalis, but the insect Ankyrins contain none. Our data indicate that an ancestral Ankyrin acquired a 18 amino acid module which was preserved in the ecdysozoa/deuterostome divide, but it was subsequently lost from arthropods. Successive duplications of the module led to a gain of function in vertebrates as it acquired obscurin/titin binding activity. We suggest that the OTBD represents an adaptation of the cytoskeleton that confers muscle cells with resilience to the forces associated with vertebrate life.

  • Ankyrin-G in skeletal muscle: Tissue-specific alternative splicing contributes to the complexity of the sarcolemmal cytoskeleton.
    Experimental Cell Research, 2005
    Co-Authors: Alexander Hopitzan, Anthony Baines, Marie Aline Ludosky, Michel Recouvreur, Ekaterini Kordeli
    Abstract:

    Ankyrins are versatile adaptor proteins that join the spectrin-based cytoskeleton to transmembrane proteins, and have roles in organizing the microstructure of cell membranes. Molecular diversity of Ankyrins in mammals arises from extensive alternative splicing of the products of three genes. There has been no systematic analysis of the diversity of expression of Ankyrins-G, the widely expressed Ank3 gene products, in a complex tissue. We previously described AnkG107, the first muscle-specific Ankyrin-G. Here we combined cDNA and database analyses to gain novel insight into the Ankyrins-G of skeletal muscle. We find: (i) that Ank3 is composed of at least 53 exons, many of which are subject to tissue-specific splicing; (ii) five novel full-length cDNAs encoding two canonical (AnkG197, AnkG217) and three small isoforms (AnkG109, AnkG128, AnkG130) bring to six the number of Ankyrins-G expressed in skeletal muscle; (iii) a 76-residue C-terminal domain is a ‘signature' for muscle Ankyrins; (iv) variably spliced sequences of 17/18 and 195 residues increase diversity in the C-terminal domains. Comparison of endogenous Ankyrins-G with in vitro translated cDNAs revealed that small Ankyrins account for the majority of the immunoreactivity for Ankyrin-G in soleus muscle. The small Ankyrins, when expressed in vivo in the rat muscle, are all targeted to sarcolemmal costameres. Our results demonstrate the tissue-dependent alternative splicing of Ank3 in skeletal muscle and point to novel functions of small Ankyrins-G in organizing microdomains of the plasma membrane.

  • Identification of Ank G107 , a Muscle-specific Ankyrin-G Isoform
    Journal of Biological Chemistry, 2002
    Co-Authors: Claire Gagelin, Jean Cartaud, Marie Aline Ludosky, Christiane Deprette, Michel Recouvreur, Bruno Constantin, Christian Cognard, Guy Raymond, Ekaterini Kordeli
    Abstract:

    We previously showed that alternatively spliced Ankyrins-G, the Ank3 gene products, are expressed in skeletal muscle and localize to the postsynaptic folds and to the sarcoplasmic reticulum. Here we report the molecular cloning, tissue expression, and subcellular targeting of Ank(G107), a novel Ankyrin-G from rat skeletal muscle. Ank(G107) lacks the entire ANK repeat domain and contains a 76-residue sequence near the COOH terminus. This sequence shares homology with COOH-terminal sequences of Ankyrins-R and Ankyrins-B, including the muscle-specific skAnk1. Despite widespread tissue expression of Ank3, the 76-residue sequence is predominantly detected in transcripts of skeletal muscle and heart, including both major 8- and 5.6-kb mRNAs of skeletal muscle. In 15-day-old rat skeletal muscle, antibodies against the 76-residue sequence localized to the sarcolemma and to the postsynaptic membrane and cross-reacted with three endogenous Ankyrins-G, including one 130-kDa polypeptide that comigrated with in vitro translated Ank(G107). In adult muscle, these polypeptides appeared significantly decreased, and immunofluorescence labeling was no more detectable. Green fluorescent protein-tagged Ank(G107) transfected in primary cultures of rat myotubes was targeted to the plasma membrane. Deletion of the 76-residue insert resulted in additional cytoplasmic labeling suggestive of a reduced stability of Ank(G107) at the membrane. Recruitment of the COOH-terminal domain to the membrane was much less efficient but still possible only in the presence of the 76-residue insert. We conclude that the 76-residue sequence contributes to the localization and is essential to the stabilization of Ank(G107) at the membrane. These results suggest that tissue-dependent and developmentally regulated alternative processing of Ankyrins generates isoforms with distinct sequences, potentially involved in specific protein-protein interactions during differentiation of the sarcolemma and, in particular, of the postsynaptic membrane.

  • AnkyrinG is associated with the postsynaptic membrane and the sarcoplasmic reticulum in the skeletal muscle fiber
    Journal of Cell Science, 1998
    Co-Authors: Ekaterini Kordeli, Marie Aline Ludosky, Christiane Deprette, Thierry Frappier, Jean Cartaud
    Abstract:

    Ankyrins are a multi-gene family of peripheral proteins that link ion channels and cell adhesion molecules to the spectrin-based skeleton in specialized membrane domains. In the mammalian skeletal myofiber, Ankyrins were immunolocalized in several membrane domains, namely the costameres, the postsynaptic membrane and the triads. Ank1 and Ank3 transcripts were previously detected in skeletal muscle by northern blot analysis. However, the Ankyrin isoforms associated with these domains were not identified, with the exception of an unconventional Ank1 gene product that was recently localized at discrete sites of the sarcoplasmic reticulum. Here we study the expression and subcellular distribution of the Ank3 gene products, the AnkyrinsG, in the rat skeletal muscle fiber. Northern blot analysis of rat skeletal muscle mRNAs using domain-specific Ank3 cDNA probes revealed two transcripts of 8.0 kb and 5.6 kb containing the spectrin-binding and C-terminal, but not the serine-rich, domains. Reverse transcriptase PCR analysis of rat skeletal muscle total RNA confirmed the presence of Ank3 transcripts that lacked the serine-rich and tail domains, a major insert of 7813 bp at the junction of the spectrin-binding and C-terminal domains that was previously identified in brain Ank3 transcripts. Immunoblot analysis of total skeletal muscle homogenates using AnkyrinG-specific antibodies revealed one major 100 kDa AnkyrinG polypeptide. Immunofluorescence labeling of rat diaphragm cryosections showed that Ankyrin(s)G are selectively associated with (1) the depths of the postsynaptic membrane folds, where the voltage-dependent sodium channel and N-CAM accumulate, and (2) the sarcoplasmic reticulum, as confirmed by codistribution with the sarcoplasmic reticulum Ca2+-ATPase (SERCA 1). At variance with Ankyrin(s)G, Ankyrin(s)R (ank1 gene products) accumulate at the sarcolemma and at sarcoplasmic structures, in register with A-bands. Both Ankyrin isoforms codistributed over Z-lines and at the postsynaptic membrane. These data extend the notion that Ankyrins are differentially localized within myofibers, and point to a role of the AnkyrinG family in the organization of the sarcoplasmic reticulum and the postsynaptic membrane.

  • Ankyring a new Ankyrin gene with neural specific isoforms localized at the axonal initial segment and node of ranvier
    Journal of Biological Chemistry, 1995
    Co-Authors: Ekaterini Kordeli, Stephen Lambert, Vann Bennett
    Abstract:

    We have characterized a new Ankyrin gene, expressed in brain and other tissues, that is subject to extensive tissue-specific alternative mRNA processing. The full-length polypeptide has a molecular mass of 480 kDa and includes a predicted globular head domain, with membrane- and spectrin-binding activities, as well as an extended "tail" domain. We term this gene AnkyrinG based on its giant size and general expression. Two brain-specific isoforms of 480 kDa and 270 kDa were identified that contain a unique stretch of sequence highly enriched in serine and threonine residues immediately following the globular head domain. Antibodies against the serine-rich domain and spectrin-binding domain revealed labeling of nodes of Ranvier and axonal initial segments. Ankyrin-binding proteins also known to be localized in these specialized membrane domains include the voltage-dependent sodium channel, the sodium/potassium ATPase, sodium/calcium exchanger, and members of the neurofascin/L1 family of cell adhesion molecules. The neural-specific AnkyrinG polypeptides are candidates to participate in maintenance/targeting of ion channels and cell adhesion molecules to nodes of Ranvier and axonal initial segments.

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

  • the campoletis sonorensis ichnovirus vAnkyrin protein p vank 1 inhibits apoptosis in insect sf9 cells
    Insect Molecular Biology, 2009
    Co-Authors: Angelika Fathgoodin, Jeremy A Kroemer, Bruce A. Webb
    Abstract:

    : The Campoletis sonorensis ichnovirus (CsIV) vAnkyrin genes encode proteins containing truncated Ankyrin repeat domains with sequence homology to the inhibitory domains of NF-kappaB transcription factor inhibitors, IkappaBs. The CsIV vAnkyrin proteins are thought to be involved in the suppression of NF-kappaB activity during immune response and/or developmental events in the parasitized host. Here we report that when P-vank-1 was expressed stably from Sf9 cells, prolonged survival of these cells was observed after baculovirus infection, UV irradiation, and treatment with the apoptosis-inducing chemical camptothecin compared to untransformed Sf9 cells. Furthermore, P-vank-1 inhibited nuclear and internucleosomal degradation and caspase activity after induction of apoptosis in Sf9 cells stably expressing P-vank-1. This is the first report of a polydnavirus protein with anti-apoptotic function.

  • Divergences in protein activity and cellular localization within the Campoletis sonorensis Ichnovirus VAnkyrin family.
    Journal of virology, 2006
    Co-Authors: Jeremy A Kroemer, Bruce A. Webb
    Abstract:

    Ichnoviruses (IVs) occur in obligate symbiotic associations with endoparasitic ichneumonid wasps. IVs are injected with eggs during parasitization, where viral infection and gene expression alter host physiology to ensure endoparasitoid survival. The seven Campoletis sonorensis IV (CsIV) vAnkyrin genes encode proteins that possess Ankyrin repeat domains resembling the inhibitory domains of NF-κB transcription factor inhibitors (IκBs). The CsIV vAnkyrins are divided into two subclasses: those expressed primarily in the host fat body (three genes) and those expressed in host hemocytes (four genes). CsIV vAnkyrin proteins showed limited antigenic similarity when analyzed by Western blotting. Cellular localization and expression patterns of recombinant vAnkyrin proteins in High Five and Sf9 insect cells differed within and between the subclasses and in cells exposed to lipopolysaccharide, laminarin, or viral immune challenge. In unstimulated Sf9 cells, five vAnkyrins were detected in cell nuclei. The remaining two proteins localized predominantly to cytoplasmic granules. Immune stimulation of cells resulted in a nuclear-to-cytoplasmic shift of three vAnkyrins but did not affect localization of other variants. When expressed from recombinant Autographa californica multiple nucleopolyhedroviruses (AcMNPVs), all vAnkyrins showed a nuclear localization during early stages of infection with patterns resembling those of immune-challenged cells as the infection progressed. Two fat body vAnkyrins also produced unique biological effects when expressed from recombinant AcMNPV. Insect cells infected with these viruses exhibited enhanced longevity compared to those infected with viruses expressing other vAnkyrins. Together, these data suggest that vAnkyrin proteins in CsIV have divergent physiological functions.

  • Iκβ-Related vAnkyrin Genes in the Campoletis sonorensis Ichnovirus: Temporal and Tissue-Specific Patterns of Expression in Parasitized Heliothis virescens Lepidopteran Hosts
    Journal of virology, 2005
    Co-Authors: Jeremy A Kroemer, Bruce A. Webb
    Abstract:

    Polydnaviruses (PDVs) are unusual insect viruses that occur in obligate symbiotic associations with parasitic ichneumonid (ichnoviruses, or IVs) and braconid (bracoviruses, or BVs) wasps. PDVs are injected with eggs, ovarian proteins, and venom during parasitization. Following infection of cells in host tissues, viral genes are expressed and their products function to alter lepidopteran host physiology, enabling endoparasitoid development. Here we describe the Campoletis sonorensis IV viral Ankyrin (vAnkyrin) gene family and its transcription. The seven members of this gene family possess Ankyrin repeat domains that resemble the inhibitory domains of the Drosophila melanogaster NF-κβ transcription factor inhibitor (Iκβ) cactus. vAnkyrin gene expression is detected within 2 to 4 h postparasitization (p.p.) in Heliothis virescens hosts and reaches peak levels by 3 days p.p. Our data indicate that vAnkyrin genes from the C. sonorensis IV genome are differentially expressed in the tissues of parasitized hosts and can be divided into two subclasses: those that target the host fat body and those that target host hemocytes. Polyclonal antibodies raised against a fat-body targeting vAnkyrin detected a 19-kDa protein in crude extracts prepared from the 3 days p.p. fat body. VAnkyrin-specific Abs localized to 3-day p.p. fat-body and hemocyte nuclei, suggesting a role for vAnkyrin proteins in the nuclei of C. sonorensis IV-infected cells. These data are evidence for divergent tissue specificities and targeting of multigene families in IVs. We hypothesize that PDV vAnkyrin genes may suppress NF-κβ activity during immune responses and developmental cascades in parasitized lepidopteran hosts of C. sonorensis.