The necdin interactome: evaluating the effects of amino acid substitutions and cell stress using proximity-dependent bio
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ORIGINAL INVESTIGATION
The necdin interactome: evaluating the effects of amino acid substitutions and cell stress using proximity‑dependent biotinylation (BioID) and mass spectrometry Matthea R. Sanderson1 · Katherine E. Badior2,3 · Richard P. Fahlman2,4 · Rachel Wevrick1 Received: 21 February 2020 / Accepted: 3 June 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Prader–Willi syndrome (PWS) is a neurodevelopmental disorder caused by the loss of function of a set of imprinted genes on chromosome 15q11–15q13. One of these genes, NDN, encodes necdin, a protein that is important for neuronal differentiation and survival. Loss of Ndn in mice causes defects in the formation and function of the nervous system. Necdin is a member of the melanoma-associated antigen gene (MAGE) protein family. The functions of MAGE proteins depend highly on their interactions with other proteins, and in particular MAGE proteins interact with E3 ubiquitin ligases and deubiquitinases to form MAGE-RING E3 ligase-deubiquitinase complexes. Here, we used proximity-dependent biotin identification (BioID) and mass spectrometry (MS) to determine the network of protein–protein interactions (interactome) of the necdin protein. This process yielded novel as well as known necdin-proximate proteins that cluster into a protein network. Next, we used BioID-MS to define the interactomes of necdin proteins carrying coding variants. Variant necdin proteins had interactomes that were distinct from wildtype necdin. BioID-MS is not only a useful tool to identify protein–protein interactions, but also to analyze the effects of variants of unknown significance on the interactomes of proteins involved in genetic disease. Abbreviations BioID Proximity-dependent biotin identification LC–MS/MS Liquid chromatography–tandem mass spectrometry MAGE Melanoma antigen gene MHD MAGE homology domain MS Mass spectrometry PWS Prader–Willi syndrome WH Winged helix WT Wildtype Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00439-020-02193-9) contains supplementary material, which is available to authorized users. * Rachel Wevrick [email protected] 1
Department of Medical Genetics, University of Alberta, Edmonton, AB, Canada
2
Department of Biochemistry, University of Alberta, Edmonton, AB, Canada
3
Membrane Protein Disease Research Group, University of Alberta, Edmonton, AB, Canada
4
Department of Oncology, University of Alberta, Edmonton, AB, Canada
Introduction Prader–Willi syndrome (PWS) is a neurodevelopmental disorder caused by the loss of function of a set of imprinted genes on chromosome 15q11–15q13. PWS is characterized by hyperphagia, hypotonia, endocrine dysfunction, intellectual disability, and autism spectrum disorder. One of the genes inactivated in PWS, NDN, encodes necdin, a 321 amino acid melanoma-associated antigen (MAGE) family protein. MAGE proteins interact with E3 ubiquitin ligases and deubiquitinases to form MAGE-RING E3 ligase complexes. These comple
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