Distinct functional domains in nesprin-1alpha and nesprin-2beta bind directly to emerin and both interactions are disrupted in X-linked Emery-Dreifuss muscular dystrophy.
ABSTRACT Emerin and specific isoforms of nesprin-1 and -2 are nuclear membrane proteins which are binding partners in multi-protein complexes spanning the nuclear envelope. We report here the characterisation of the residues both in emerin and in nesprin-1alpha and -2beta which are involved in their interaction and show that emerin requires nesprin-1 or -2 to retain it at the nuclear membrane. Using several protein-protein interaction methods, we show that residues 368 to 627 of nesprin-1alpha and residues 126 to 219 of nesprin-2beta, which show high homology to one another, both mediate binding to emerin residues 140-176. This region has previously been implicated in binding to F-actin, beta-catenin and lamin A/C suggesting that it is critical for emerin function. Confirmation that these protein domains interact in vivo was shown using GFP-dominant negative assays. Exogenous expression of either of these nesprin fragments in mouse myoblast C2C12 cells displaced endogenous emerin from the nuclear envelope and reduced the targeting of newly synthesised emerin. Furthermore, we are the first to report that emerin mutations which give rise to X-linked Emery-Dreifuss muscular dystrophy, disrupt binding to both nesprin-1alpha and -2beta isoforms, further indicating a role of nesprins in the pathology of Emery-Dreifuss muscular dystrophy.
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ABSTRACT: Nuclear envelope spectrin-repeat proteins (Nesprins), are a novel family of nuclear and cytoskeletal proteins with rapidly expanding roles as intracellular scaffolds and linkers. Originally described as proteins that localise to the nuclear envelope (NE) and establish nuclear-cytoskeletal connections, nesprins have now been found to comprise a diverse spectrum of tissue specific isoforms that localise to multiple sub-cellular compartments. Here, we describe how nesprins are necessary in maintaining cellular architecture by acting as essential scaffolds and linkers at both the NE and other sub-cellular domains. More importantly, we speculate how nesprin mutations may disrupt tissue specific nesprin scaffolds and explain the tissue specific nature of many nesprin-associated diseases, including laminopathies.Expert Reviews in Molecular Medicine 01/2013; 15:e5. · 6.62 Impact Factor
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ABSTRACT: Nesprin-1 and nesprin-2 are nuclear envelope (NE) proteins characterized by a common structure of an SR (spectrin repeat) rod domain and a C-terminal transmembrane KASH [Klarsicht-ANC-Syne-homology] domain and display N-terminal actin-binding CH (calponin homology) domains. Mutations in these proteins have been described in Emery-Dreifuss muscular dystrophy and attributed to disruptions of interactions at the NE with nesprins binding partners, lamin A/C and emerin. Evolutionary analysis of the rod domains of the nesprins has shown that they are almost entirely composed of unbroken SR-like structures. We present a bioinformatical approach to accurate definition of the boundaries of each SR by comparison with canonical SR structures, allowing for a large-scale homology modelling of the 74 nesprin-1 and 56 nesprin-2 SRs. The exposed and evolutionary conserved residues identify important pbs for protein-protein interactions that can guide tailored binding experiments. Most importantly, the bioinformatics analyses and the 3D models have been central to the design of selected constructs for protein expression. 1D NMR and CD spectra have been performed of the expressed SRs, showing a folded, stable, high content α-helical structure, typical of SRs. Molecular Dynamics simulations have been performed to study the structural and elastic properties of consecutive SRs, revealing insights in the mechanical properties adopted by these modules in the cell.PLoS ONE 01/2013; 8(5):e63633. · 3.53 Impact Factor
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ABSTRACT: The nuclear lamina is a filamentous network that underlies the nuclear envelope. Lamina components include the family of LEM domain (LEM-D) proteins, named for LAP2, emerin and MAN1. Mutations in genes encoding LEM-D proteins cause tissue-restricted human disease, even though these genes are globally expressed. To understand the contributions of the LEM-D proteins to nuclear lamina function, investigations of the Drosophila LEM-D proteins was undertaken. The Drosophila genome encodes four LEM-D proteins and this thesis describes work done on the Drosophila homologues of MAN1 and emerin, Drosophila MAN1 (dMAN1) and Otefin (Ote). Chapter 2 describes the generation and phenotypic analyses of dMAN1 mutants. These mutants display a range of tissue-specific defects associated with an increase in BMP/Dpp signaling. This suggests that dMAN1 downregulates BMP/Dpp signaling at the nuclear periphery. Chapter 3 describes the identification and phenotypic analyses of ote mutants. Loss of Ote is associated with a tissue-specific defect of the female germline where ote mutant females display defects in germline stem cell (GSC) maintenance. Loss of Ote causes defects in the germline cells, the cap cells of GSC niche and an increased sensitivity to Dpp signaling in both germline and somatic cells. These findings support models suggesting that laminopathies arise from dysfunction of the homeostasis in stem cell populations. Taken together, these studies suggest that the nuclear lamina may play tissue-specific roles through regulation of signal transduction pathways. Our data also support the use of Drosophila as a system to elucidate the mechanistic basis of diseases associated with defects in the nuclear lamina.Theses and Dissertations.