Towards understanding the phosphorylation code of tau.

We describe our efforts to combine in vitro enzymatic reactions with recombinant kinases to phosphorylate the neuronal tau protein, and NMR spectroscopy to unravel the resulting phosphorylation pattern in both qualitative and quantitative manners. This approach, followed by functional assays with the same samples, gives access to the complex phosphorylation code of tau. As a result, we propose a novel hypothesis for the link between tau (hyper)phosphorylation and aggregation.

Alzheimer disease specific phosphoepitopes of Tau interfere with assembly of tubulin but not binding to microtubules.

In Alzheimer disease (AD)-affected neurons, the Tau protein is found in an aggregated and hyperphosphorylated state. A common hypothesis is that Tau hyperphosphorylation causes its dissociation from the microtubular surface, with consequently a breakdown of the microtubules (MTs) and aggregation of the unbound Tau. We evaluated the effect of Tau phosphorylation on both tubulin assembly and MT binding. We show that the cyclin-dependent kinase 2/cyclin A3 kinase complex can generate the AT8 and AT180 AD-specific phospho-epitopes and use NMR spectroscopy to validate qualitatively and quantitatively the phospho content of our samples. The simultaneous presence of both epitopes disables the tubulin assembly capacity of Tau in conditions whereby Tau is the driving force for the assembly process but does not, however, inhibit MT assembly when the latter is driven by an increased tubulin concentration. When compared to the isolated MT binding repeats (K(d)=0.3 microM), the phospho-Tau retains a substantial affinity for preformed MTs (K(d)=11 nM), suggesting that the phosphorylated proline-rich region still participates in the binding event. Our results hence indicate that the sole phosphorylation at the AT8/AT180 epitopes, although leading to a functional defect for Tau, is not sufficient for its dissociation from the MT surface and subsequent aggregation as observed in AD.

Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy.

A series of fluorophores with single-exponential fluorescence decays in liquid solution at 20 degrees C were measured independently by nine laboratories using single-photon timing and multifrequency phase and modulation fluorometry instruments with lasers as excitation source. The dyes that can serve as fluorescence lifetime standards for time-domain and frequency-domain measurements are all commercially available, are photostable under the conditions of the measurements, and are soluble in solvents of spectroscopic quality (methanol, cyclohexane, water). These lifetime standards are anthracene, 9-cyanoanthracene, 9,10-diphenylanthracene, N-methylcarbazole, coumarin 153, erythrosin B, N-acetyl-l-tryptophanamide, 1,4-bis(5-phenyloxazol-2-yl)benzene, 2,5-diphenyloxazole, rhodamine B, rubrene, N-(3-sulfopropyl)acridinium, and 1,4-diphenylbenzene. At 20 degrees C, the fluorescence lifetimes vary from 89 ps to 31.2 ns, depending on fluorescent dye and solvent, which is a useful range for modern pico- and nanosecond time-domain or mega- to gigahertz frequency-domain instrumentation. The decay times are independent of the excitation and emission wavelengths. Dependent on the structure of the dye and the solvent, the excitation wavelengths used range from 284 to 575 nm, the emission from 330 to 630 nm. These lifetime standards may be used to either calibrate or test the resolution of time- and frequency-domain instrumentation or as reference compounds to eliminate the color effect in photomultiplier tubes. Statistical analyses by means of two-sample charts indicate that there is no laboratory bias in the lifetime determinations. Moreover, statistical tests show that there is an excellent correlation between the lifetimes estimated by the time-domain and frequency-domain fluorometries. Comprehensive tables compiling the results for 20 (fluorescence lifetime standard/solvent) combinations are given.

NMR investigation of the interaction between the neuronal protein tau and the microtubules.

Whereas the interaction between Tau and the microtubules has been studied in great detail both by macroscopic techniques (cosedimentation, cryo-electron microscopy, and fluorescence spectroscopy) using the full-length protein or by peptide mapping assays, no detailed view at the level of individual amino acids has been presented when using the full-length protein. Here, we present a nuclear magnetic resonance (NMR) study of the interaction between the full-length neuronal protein Tau and paclitaxel-stabilized microtubules (MTs). As signal disappearance in the heteronuclear 1H-15N correlation spectra of isotope-labeled Tau in complex with MTs is due to direct association of the corresponding residue with the solid-like MT wall, we can map directly the fragment in interaction with the MT surface, and obtain a molecular picture of the precise interaction zones. The N-terminal region projects from the microtubule surface, and the lack of chemical shift variations when compared with free Tau proves that this region can regulate microtubular separation without adopting a stable conformation. Amino acids in the four microtubule binding repeats (MTBRs) lose all of their intensity, underscoring their immobilization upon binding to the MTs. The same loss of NMR intensity was observed for the proline-rich region starting at Ser214, underscoring its importance in the Tau:MT interaction. Fluorescence resonance energy transfer (FRET) experiments were used to obtain thermodynamic binding parameters, and led to the conclusion that the NMR defined fragment indeed is the major player in the interaction. When the same Ser214 is phosphorylated by the PKA kinase, the Tau:MT interaction strength decreases by 2 orders of magnitude, but the proline-rich region including the phospho-Ser214 does not gain sufficient mobility in the complex to make it observable by NMR spectroscopy. The presence of an intramolecular disulfide bridge, on the contrary, does lead to a partial detachment of the C-terminus of Tau, and decreases significantly the overloading of Tau on the MT surface.

Tau aggregation in Alzheimer's disease: what role for phosphorylation?

The crucial role of the neuronal Tau protein in microtubule stabilization and axonal transport suggests that too little or too much Tau might lead to neuronal dysfunction. The presence of a hyper phosphorylated but non aggregated molecule as a toxic species that might sequester normal Tau is discussed. We present recent in vitro results that might allow us to dissect the role of individual phosphorylation sites on its structure and function. We also discuss in this review the role of phosphorylation for the aggregation of the neuronal Tau protein, and compare it to the aggregation induced by external poly anions.

Structural impact of heparin binding to full-length Tau as studied by NMR spectroscopy.

The neuronal Tau protein is involved in stabilizing microtubules but is also the major component of the paired helical filaments (PHFs), the intracellular aggregates that characterize Alzheimer's disease (AD) in neurons. In vitro, Tau can be induced to form AD-like aggregates by adding polyanions such as heparin. While previous studies have identified the microtubule binding repeats (MTBRs) as the major player in Tau aggregation, the fact that the full-length protein does not aggregate by itself indicates the presence of inhibitory factors. Charge and conformational changes are of uttermost importance near the second (R2) and third (R3) MTBR that are thought to be involved directly in the nucleation of the aggregation. Recently, the positively charged regions flanking the MTBR were proposed to inhibit PHF assembly, where hyperphosphorylation neutralizes these basic inhibitory domains, enabling Tau-Tau interactions. Here we present results of an NMR study on the interaction between intact full-length Tau and small heparin fragments of well-defined size, under conditions where no aggregation occurs. Our findings reveal (i) micromolar affinity of heparin to residues in R2 and R3, (ii) two zones of strong interaction within the positively charged inhibitory regions flanking the MTBR, and (iii) another interaction site upstream of the two inserts encoded by exons 2 and 3. Three-dimensional heteronuclear NMR experiments demonstrate that the interaction with heparin induces beta-strand structure in several regions of Tau that might act as nucleation sites for its aggregation but indicate as well alpha-helical structure in regions outside the core of PHF. In the PHF, the residues outside of the core maintain sufficient mobility for NMR detection and recover their unbound chemical shift values after an overnight incubation at 37 degrees C with heparin. Heparin thus becomes integrated into the rigid core region of the PHF, probably providing the charge compensation for the lysine-rich stretches that form upon the in-register, parallel stacking of the repeat regions.

Studying the natively unfolded neuronal Tau protein by solution NMR spectroscopy.

The neuronal Tau protein, whose physiological role is to stabilize the microtubules, is found under the form of aggregated filaments and tangles in Alzheimer's diseased neurons. Until recently detailed structural analysis of the natively unfolded Tau protein has been hindered due to its shear size and unfavourable amino acid composition. We review here the recent progress in the assignments of the full-length polypeptide using novel methods of product planes and peptide NMR mapping, and indicate the structural insights that can be obtained from this assignment. Preliminary NMR data on the fibers show that the assignment enables a precise mapping of the rigid core. Future NMR experiments should allow one to gain more insight into the conformational aspects of this intriguing protein.

NMR analysis of a Tau phosphorylation pattern.

The phosphorylation of the neuronal Tau protein modulates both its physiological role of microtubule binding and its aggregation into paired helical fragments observed in Alzheimer's diseased neurons. However, detailed knowledge of the role of phosphorylation at specific sites has been hampered by the analytical difficulties to evaluate the level of site-specific phosphate incorporation. Even with recombinant kinases, mass spectrometry and immunodetection are not evident for determining the full phosphorylation pattern in a qualitative and quantitative manner. We show here that heteronuclear NMR spectroscopy on a 15N labeled Tau sample modified by the cAMP dependent kinase allows identification of all phosphorylation sites, measures their level of phosphate integration, and yields kinetic data for the enzymatic modification of the individual sites. Filtering through the 15N label discards the necessity of any further sample purification and allows the in situ monitoring of kinase activity at selected sites. We finally demonstrate that the NMR approach can equally be used to evaluate potential kinase inhibitors in a straightforward manner.

Regions of tau implicated in the paired helical fragment core as defined by NMR.

We have studied the mature Alzheimer-like fibers of tau by fluorescence and NMR spectroscopy. Assembly of the protein into paired helical filaments after incubation with heparin at 37 degrees C was verified by electron microscopy and size-exclusion chromatography. NMR spectroscopy on these mature fibers revealed different regions of residual mobility for tau: the N-terminal domain was found to maintain solution-like dynamics and was followed by a large domain of decreasing mobility; finally the core region was distinguished by a solid-like character. Heteronuclear-NOE data indicate that the decreasing mobility is due to both a slowing down of the rapid nanosecond movements and the introduction of slower movements that lead to exchange broadening. Fluorescence spectroscopy confirmed the presence of this rigid core, and some degree of protection from hydrogen exchange for those residues was observed. Hence, our data give a more precise picture of the dynamics of tau when it is integrated into mature filaments and should provide further understanding of the molecular processes that govern aggregation.

High-resolution magic angle spinning NMR of the neuronal tau protein integrated in Alzheimer's-like paired helical fragments.

HRMAS NMR of tau paired helical fragments assembled with heparin show an intensity decrease for those amino acids that are incorporated into the rigid core region, whereas the N-terminal amino acids maintain their full mobility.

Accepting its random coil nature allows a partial NMR assignment of the neuronal Tau protein.

A combined strategy to obtain a partial NMR assignment of the neuronal Tau protein is presented. Confronted with the extreme spectral degeneracy that the spectrum of this 441 amino acid long unstructured protein presents, we have introduced a graphical procedure based on residue type-specific product planes. Combining this strategy with the search for pairwise motifs, and combining the spectra of different Tau isoforms and even of peptides derived from the native sequence, we arrive at a partial assignment that is sufficient to map the interactions of Tau with its molecular partners. The obtained assignments equally confirm the absence of regular secondary structure in the isolated protein.

Proline-directed random-coil chemical shift values as a tool for the NMR assignment of the tau phosphorylation sites.

NMR spectroscopy of the full-length neuronal Tau protein has proved to be difficult due to the length of the protein and the unfavorable amino acid composition. We show that the random-coil chemical shift values and their dependence on the presence of a proline residue in the (i+1) position can successfully be exploited to assign all proline-directed phosphorylation sites. This is a first step toward the study of the phosphorylation of Tau by NMR spectroscopy.

Mechanism of fluorescence and conformational changes of the sarcoplasmic calcium binding protein of the sand worm Nereis diversicolor upon Ca2+ or Mg2+ binding.

The calcium-binding protein isolated from the sarcoplasm of the muscles of the sand worm Nereis diversicolor has four EF-hands and three active binding sites for Ca(2+) or Mg(2+). Nereis diversicolor sarcoplasmic calcium-binding protein contains three tryptophan residues at positions 4, 57, and 170, respectively. The Wt protein shows a very limited fluorescence increase upon binding of Ca(2+) or Mg(2+). Single-tryptophan-containing mutants were produced and purified. The fluorescence titrations of these mutants show a limited decrease of the affinity for calcium, but no alterations of the cooperativity. Upon adding calcium, Trp170 shows a strong fluorescence increase, Trp57 an extensive fluorescence decrease, and Trp4 shows no fluorescence change. Therefore mutant W4F/W170F is ideally suited to analyze the fluorescence titrations and to study the binding mechanism. Mutations of the calcium ligands at the z-position in the three binding sites show no effect at site I and a total loss of cooperativity at sites III and IV. The quenching of Trp57 upon calcium binding is dependent on the presence of arginine R25, but this residue is not just a simple dynamic quencher. The role of the salt bridge R25-D58 is also investigated.

Tryptophan properties in fluorescence and functional stability of plasminogen activator inhibitor 1.

Plasminogen activator inhibitor 1 harbors four tryptophan residues at positions 86, 139, 175, and 262. To investigate the contribution of each tryptophan residue to the total fluorescence and to reveal the mutual interactions of the tryptophan residues and interactions with the other amino acids, 15 mutants in which tryptophan residues have been replaced by phenylalanines were constructed, purified, and characterized. Conformational distribution analysis revealed that the tryptophan mutants have a similar conformational distribution pattern as wild-type plasminogen activator inhibitor 1. Mutants in which tryptophan residue 175 was replaced by a phenylalanine displayed an increased functional half-life of the active conformation, whereas the functional half-life of mutants in which tryptophan residue 262 was replaced by a phenylalanine was substantially decreased. Comparative analysis of the fluorescence lifetimes, the extinction coefficients, and the quantum yields of the individual tryptophan residues demonstrates that tryptophan residue 262 gives the highest contribution to the total fluorescence. The other tryptophan residues have a very low quantum yield. In the wild-type protein, the fluorescence of all tryptophan residues is partially quenched as compared to the mutants that contain single tryptophan residues, due to conformational effects. The fluorescence of tryptophan residue 262 is very likely also partially quenched by energy transfer to tryptophan residue 175.