The interactions of neuronal phospho-protein Tau with its protein partners as therapeutic targets in Alzheimer’s disease

Tau est une protéine neuronale qui est observé hyperphosphorylé et agrégée dans les filaments hélicaux appariés (PHFs) dans les cerveaux de la maladie d’Alzheimer. La spectroscopie de résonance magnétique nucléaire a permis une caractérisation analytique de la protéine Tau phosphorylée, également d’étudier les interactions de Tau avec des partenaires moléculaires. Parmi de 16 motifs pS/pT-P présents dans la séquence de Tau, il y a 14 sites phosphorylés par ERK2(Extracellular signal-Regulated Kinase2). Ce profil de phosphorylation est très semblable à celui obtenu avec un extrait de cerveau de rat. En plus, il est montré que la protéine Tau phosphorylée uniquement par ERK2 présente des propriétés d’agrégation semblables à la protéine Tau phosphorylée par les extraits de cerveaux de rat. Nous avons mis en évidence que Tau est un substrat de ERK2 reconnu par des sites multiples d’ancrage. Par ailleurs, des oligonucléotides interagissent avec Tau au niveau des séquences [209-246] et [267-289], et cette interaction est abolie par la phosphorylation de Tau par la kinase ERK. En conclusion, la phosphorylation de Tau par ERK sur les motifs S/T-P, localisés tout le long de sa séquence mais particulièrement dans le domaine régulateur riche en proline, a un impact sur les capacités d’agrégation de la protéine modifiée et sur ses propriétés d’interaction avec un partenaire moléculaire physiologique, l’ADN. Ces résultats concordent avec les études de réalisées dans des contextes cellulaires qui identifient ERK comme une kinase activée dans des conditions de stress du neurone qui pourrait conduire à une phosphorylation pathologique de Tau.Tau has a central role in neurodegeneration and is implicated in Alzheimer’s disease development. It is found aggregated in neurons affected by the disease, typically in paired helical filaments (PHFs) constituted of hyper-phosphorylated Tau. Nuclear Magnetic resonance Spectroscopy is used to characterize Tau phosphorylation pattern, as well as to study Tau interactions with molecular partners. Analysis of in vitro phosphorylated Tau by activated recombinant ERK2(Extracellular signal-Regulated Kinase2) with NMR spectroscopy revealed phosphorylation of 14 S/T-P sites among 16 such motifs, which has a similar phosphorylation pattern In vitro by rat brain extract, and both of phosphorylated Tau show similar ability to produce pTau filaments. In addition, Tau is ERK2 substrate to present multiple docking sites instead of one high affinity single D-site motif. Additionally, I have investigated the functional consequences of Tau interaction by ERK. Oligonucleotide sequences interact with [209-246] and [267-289] regions. I have shown that this interaction is abolished by Tau phosphorylation by ERK2. This study shows that the ERK2 kinase has the capacity by itself to phosphorylate Tau on many sites and that the resulting phosphorylation pattern increases pTau aggregation propensity. Moreover, ERK2 phosphorylation of Tau can lead to a loss of physiological function, such as its capacity to bind DNA. These results support the hypothesis that ERK activation might have a detrimental effect for Tau function and participate in AD physio-pathology

NMR meets Tau: insights into its function and pathology

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

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

International audienceThe 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

Identification of the Tau phosphorylation pattern that drives its aggregation

International audienceDetermining the functional relationship between Tau phosphorylation and aggregation has proven a challenge owing to the multiple potential phosphorylation sites and their clustering in the Tau sequence. We use here in vitro kinase assays combined with NMR spectroscopy as an analytical tool to generate well-characterized phosphorylated Tau samples and show that the combined phosphorylation at the Ser202/Thr205/Ser208 sites, together with absence of phosphorylation at the Ser262 site, yields a Tau sample that readily forms fibers, as observed by thioflavin T fluorescence and electron microscopy. On the basis of conformational analysis of synthetic phosphorylated peptides, we show that aggregation of the samples correlates with destabilization of the turn-like structure defined by phosphorylation of Ser202/Thr205

Nuclear Magnetic Resonance Analysis of the Acetylation Pattern of the Neuronal Tau Protein

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 <i>in vitro</i>. 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

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

International audienceThe immunophilin FKBP52 interacts with nuclear steroid hormone receptors. Studying the crystal structure of human estrogen receptor alpha (hER alpha) 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 beta-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

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 V³⁶⁴PGF³⁶⁷ 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 New Bis(phthalocyaninato) Terbium Single-Ion Magnet with an Overall Excellent Magnetic Performance

Bulky and strong electron-donating dibutylamino groups were incorporated onto the peripheral positions of one of the two phthalocyanine ligands in the bis­(phthalocyaninato) terbium complex, resulting in the isolation of heteroleptic double-decker (Pc)­Tb­{Pc­[N­(C₄H₉)₂]₈} {Pc = phthalocyaninate; Pc­[N­(C₄H₉)₂]₈ = 2,3,9,10,16,17,23,24-octakis­(dibutylamino)­phthalocyaninate} with the nature of an unsymmetrical molecular structure, a square-antiprismatic coordination geometry, an intensified coordination field strength, and the presence of organic radical-f interaction. As a total result of all these factors, this sandwich-type tetrapyrrole lanthanide single-ion magnet (SIM) exhibits an overall enhanced magnetic performance including a high blocking temperature (<i>T</i>_B) of 30 K and large effective spin-reversal energy barrier of <i>U</i>_eff = 939 K, rendering it the best sandwich-type tetrapyrrole lanthanide SIM reported thus far