In JoVE (1)
Articles by Clément Despres in JoVE
Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins Clément Danis*1,2, Clément Despres*1, Luiza M. Bessa1, Idir Malki1, Hamida Merzougui1, Isabelle Huvent1, Haoling Qi1, Guy Lippens1, François-Xavier Cantrelle1, Robert Schneider1, Xavier Hanoulle1, Caroline Smet-Nocca1, Isabelle Landrieu1 1UMR8576, CNRS, Lille University, 2UMR-S1172, INSERM CNRS, Lille University We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.
Other articles by Clément Despres on PubMed
Semi-synthesis of a Tag-free O-GlcNAcylated Tau Protein by Sequential Chemoselective Ligation Journal of Peptide Science : an Official Publication of the European Peptide Society. May, 2016 | Pubmed ID: 27071766 In this paper, the first semi-synthesis of the Alzheimer-relevant tau protein carrying an O-GlcNAcylation is demonstrated by using sequential chemoselective ligation. The 52-amino acid C-terminus of tau was obtained by native chemical ligation between two synthetic peptide fragments, one carrying the O-GlcNAc moiety on Ser400, which has recently been demonstrated to inhibit tau phosphorylation and to hinder tau oligomerization, and the other equipped with a photocleavable biotin handle. After desulfurization to deliver a native alanine at the ligation junction, the N-terminal cysteine was unmasked, and the peptide was further used for expressed protein ligation to generate the full-length tau protein, which was purified by a photocleavable biotin tag. We thus provide a synthetic route to obtain a homogenous tag-free O-GlcNAcylated tau protein that can further help to elucidate the significance of posttranslational modification on the tau protein and pave the way for evaluating possible drug targets in Alzheimer's disease. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
NMR Meets Tau: Insights into Its Function and Pathology Biomolecules. Jun, 2016 | Pubmed ID: 27338491 In this review, we focus on what we have learned from Nuclear Magnetic Resonance (NMR) studies on the neuronal microtubule-associated protein Tau. We consider both the mechanistic details of Tau: the tubulin relationship and its aggregation process. Phosphorylation of Tau is intimately linked to both aspects. NMR spectroscopy has depicted accurate phosphorylation patterns by different kinases, and its non-destructive character has allowed functional assays with the same samples. Finally, we will discuss other post-translational modifications of Tau and its interaction with other cellular factors in relationship to its (dys)function.
The Study of Posttranslational Modifications of Tau Protein by Nuclear Magnetic Resonance Spectroscopy: Phosphorylation of Tau Protein by ERK2 Recombinant Kinase and Rat Brain Extract, and Acetylation by Recombinant Creb-Binding Protein Methods in Molecular Biology (Clifton, N.J.). 2017 | Pubmed ID: 27975251 Nuclear magnetic resonance (NMR) spectroscopy can be used as an analytical tool to investigate posttranslational modifications of protein. NMR is a valuable tool to map the interaction regions of protein partners. Here, we present protocols that have been developed in the course of our studies of the neuronal Tau protein. Tau is found aggregated in the neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation and acetylation of Tau. We have used NMR to investigate how these posttranslational modifications of Tau affect the interactions with its partners. We present here detailed protocols of in vitro phosphorylation of Tau by recombinant kinase, ERK2, or kinase activity of rat brain extracts, and acetylation by recombinant Creb-binding protein (CBP) acetyltransferase. The analytical characterization of the modified Tau by NMR spectroscopy is additionally described.