nuclear-magnetic-resonance-spectroscopy-for-identification-multiple

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

Lille University

Lysine acetylation of the neuronal Tau protein was described as a novel mechanism of posttranslational regulation of Tau functions with important outcomes in microtubule binding and aggregation processes related to Alzheimer's disease. Here, we unravel at a per-residue resolution the acetylation pattern of full-length Tau by the Creb-binding protein (CBP) acetyltransferase using high-resolution nuclear magnetic resonance spectroscopy. Our study gives a quantitative overview of CBP-mediated acetylation and examines the catalytic proficiency because the nonenzymatic reaction with acetyl-coenzyme A occurs in vitro. Furthermore, we have investigated with this characterized acetylated Tau the effect of acetylation on Tau fibrillization in a heparin-induced aggregation assay and on heparin binding.

Nuclear magnetic resonance analysis of the acetylation pattern of the neuronal Tau protein.

The capacity of endogenous Tau to bind DNA has been recently identified in neurons under physiological or oxidative stress conditions. Characterization of the protein domains involved in Tau-DNA complex formation is an essential first step in clarifying the contribution of Tau-DNA interactions to neurological biological processes. To identify the amino acid residues involved in the interaction of Tau with oligonucleotides, we have characterized a Tau-DNA complex using nuclear magnetic resonance spectroscopy. Interaction of an AT-rich or GC-rich 22 bp oligonucleotide with Tau showed multiple points of anchoring along the intrinsically disordered Tau protein. The main sites of contact characterized here correspond to the second half of the proline-rich domain (PRD) of Tau and the R2 repeat in the microtubule binding domain. This latter interaction site includes the PHF6* sequence known to govern Tau aggregation. The characterization was pursued by studying the binding of phosphorylated forms of Tau, displaying multiple phosphorylation sites mainly in the PRD, to the same oligonucleotide. No interaction of phospho-Tau with the oligonucleotide was detected, suggesting that pathological Tau phosphorylation could affect the physiological function of Tau mediated by DNA binding.

Nuclear magnetic resonance spectroscopy characterization of interaction of Tau with DNA and its regulation by phosphorylation.

Tau neuronal protein has a central role in neurodegeneration and is implicated in Alzheimer disease development. Abnormal phosphorylation of Tau impairs its interaction with other proteins and is associated with its dysregulation in pathological conditions. Molecular mechanisms leading to hyperphosphorylation of Tau in pathological conditions are unknown. Here, we characterize phosphorylation of Tau by extracellular-regulated kinase (ERK2), a mitogen-activated kinase (MAPK) that responds to extracellular signals. Analysis ofin vitrophosphorylated Tau by activated recombinant ERK2 with nuclear magnetic resonance spectroscopy (NMR) reveals phosphorylation of 15 Ser/Thr sites.In vitrophosphorylation of Tau using rat brain extract and subsequent NMR analysis identifies the same sites. Phosphorylation with rat brain extract is known to transform Tau into an Alzheimer disease-like state. Our results indicate that phosphorylation of Tau by ERK2 alone is sufficient to produce the same characteristics. We further investigate the mechanism of ERK2 phosphorylation of Tau. Kinases are known to recognize their protein substrates not only by their specificity for a targeted Ser or Thr phosphorylation site but also by binding to linear-peptide motifs called docking sites. We identify two main ERK2 docking sites in Tau sequence using NMR. Our results suggest that ERK2 dysregulation in Alzheimer disease could lead to abnormal phosphorylation of Tau resulting in the pathology of the disease.

Characterization of Neuronal Tau Protein as a Target of Extracellular Signal-regulated Kinase.

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.

NMR Meets Tau: Insights into Its Function and Pathology.

The immunophilin FKBP52 interacts with nuclear steroid hormone receptors. Studying the crystal structure of human estrogen receptor α (hERα) and using nuclear magnetic resonance, we show here that the short V(364)PGF(367) sequence, which is located within its ligand-binding domain and adopts a type II β-turn conformation in the protein, binds the peptidyl-prolyl isomerase (PPIase or rotamase) FK1 domain of FKBP52. Interestingly, this turn motif displays strong similarities with the FKBP52 FK1 domain-binding moiety of macrolide immunomodulators such as rapamycin and GPI-1046, an immunophilin ligand with neuroprotective characteristics. An increase in the hydrophobicity of the residue preceding the proline and cyclization of the VPGF peptide strengthen its recognition by the FK1 domain of FKBP52. Replacement of the Pro residue with a dimethylproline also enhances this interaction. Our study not only contributes to a better understanding of how the interaction between the FK1 domain of FKBP52 and steroid hormone receptors most likely works but also opens new avenues for the synthesis of FKBP52 FK1 peptide ligands appropriate for the control of hormone-dependent physiological mechanisms or of the functioning of the Tau protein. Indeed, it has been shown that FKBP52 is involved in the intraneuronal dynamics of the Tau protein.

A β-Turn Motif in the Steroid Hormone Receptor's Ligand-Binding Domains Interacts with the Peptidyl-prolyl Isomerase (PPIase) Catalytic Site of the Immunophilin FKBP52.

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.

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.

Haoling Qi

UMR8576,
CNRS,
UMR8576, CNRS

Lille University

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Haoling Qi

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}UMR8576,CNRS,UMR8576, CNRS

Lille University

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

UMR8576,
CNRS,
UMR8576, CNRS