dGAE(297-391) Tau Fragment Promotes Formation of Chronic Traumatic Encephalopathy-Like Tau Filaments.

The microtubule-associated protein tau forms disease-specific filamentous aggregates in several different neurodegenerative diseases. In order to understand how tau undergoes misfolding into a specific filament type and to control this process for drug development purposes, it is crucial to study in vitro tau aggregation methods and investigate the structures of the obtained filaments at the atomic level. Here, we used the tau fragment dGAE, which aggregates spontaneously, to seed the formation of full-length tau filaments. The structures of dGAE and full-length tau filaments were investigated by magic-angle spinning (MAS) solid-state NMR, showing that dGAE allows propagation of a chronic traumatic encephalopathy (CTE)-like fold to the full-length tau. The obtained filaments efficiently seeded tau aggregation in HEK293T cells. This work demonstrates that in vitro preparation of disease-specific types of full-length tau filaments is feasible.

Shaping the future of preclinical development of successful disease-modifying drugs against Alzheimer's disease: a systematic review of tau propagation models.

The transcellular propagation of the aberrantly modified protein tau along the functional brain network is a key hallmark of Alzheimer's disease and related tauopathies. Inoculation-based tau propagation models can recapitulate the stereotypical spread of tau and reproduce various types of tau inclusions linked to specific tauopathy, albeit with varying degrees of fidelity. With this systematic review, we underscore the significance of judicious selection and meticulous functional, biochemical, and biophysical characterization of various tau inocula. Furthermore, we highlight the necessity of choosing suitable animal models and inoculation sites, along with the critical need for validation of fibrillary pathology using confirmatory staining, to accurately recapitulate disease-specific inclusions. As a practical guide, we put forth a framework for establishing a benchmark of inoculation-based tau propagation models that holds promise for use in preclinical testing of disease-modifying drugs.

Hiding in plain sight: Complex interaction patterns between Tau and 14-3-3ζ protein variants.

Tau protein is an intrinsically disordered protein that plays a key role in Alzheimer's disease (AD). In brains of AD patients, Tau occurs abnormally phosphorylated and aggregated in neurofibrillary tangles (NFTs). Together with Tau, 14-3-3 proteins - abundant cytosolic dimeric proteins - were found colocalized in the NFTs. However, so far, the molecular mechanism of the process leading to pathological changes in Tau structure as well as the direct involvement of 14-3-3 proteins are not well understood. Here, we aimed to reveal the effects of phosphorylation by protein kinase A (PKA) on Tau structural preferences and provide better insight into the interaction between Tau and 14-3-3 proteins. We also addressed the impact of monomerization-inducing phosphorylation of 14-3-3 at S58 on the binding to Tau protein. Using multidimensional nuclear magnetic resonance spectroscopy (NMR), chemical cross-linking analyzed by mass spectrometry (MS) and PAGE, we unveiled differences in their binding affinity, stoichiometry, and interfaces with single-residue resolution. We revealed that the interaction between 14-3-3 and Tau proteins is mediated not only via the 14-3-3 amphipathic binding grooves, but also via less specific interactions with 14-3-3 protein surface and, in the case of monomeric 14-3-3, also partially via the exposed dimeric interface. In addition, the hyperphosphorylation of Tau changes its affinity to 14-3-3 proteins. In conclusion, we propose quite complex interaction mode between the Tau and 14-3-3 proteins.

Conformation and Affinity Modulations by Multiple Phosphorylation Occurring in the BIN1 SH3 Domain Binding Site of the Tau Protein Proline-Rich Region.

An increase in phosphorylation of the Tau protein is associated with Alzheimer's disease (AD) progression through unclear molecular mechanisms. In general, phosphorylation modifies the interaction of intrinsically disordered proteins, such as Tau, with other proteins; however, elucidating the structural basis of this regulation mechanism remains challenging. The bridging integrator-1 gene is an AD genetic determinant whose gene product, BIN1, directly interacts with Tau. The proline-rich motif recognized within a Tau(210-240) peptide by the SH3 domain of BIN1 (BIN1 SH3) is defined as 216PTPP219, and this interaction is modulated by phosphorylation. Phosphorylation of T217 within the Tau(210-240) peptide led to a 6-fold reduction in the affinity, while single phosphorylation at either T212, T231, or S235 had no effect on the interaction. Nonetheless, combined phosphorylation of T231 and S235 led to a 3-fold reduction in the affinity, although these phosphorylations are not within the BIN1 SH3-bound region of the Tau peptide. Using nuclear magnetic resonance (NMR) spectroscopy, these phosphorylations were shown to affect the local secondary structure and dynamics of the Tau(210-240) peptide. Models of the (un)phosphorylated peptides were obtained from molecular dynamics (MD) simulation validated by experimental data and showed compaction of the phosphorylated peptide due to increased salt bridge formation. This dynamic folding might indirectly impact the BIN1 SH3 binding by a decreased accessibility of the binding site. Regulation of the binding might thus not only be due to local electrostatic or steric effects from phosphorylation but also to the modification of the conformational properties of Tau.

Biomolecular Complexation on the "Wrong Side": A Case Study of the Influence of Salts and Sugars on the Interactions between Bovine Serum Albumin and Sodium Polystyrene Sulfonate.

In the protein purification, drug delivery, food industry, and biotechnological applications involving protein-polyelectrolyte complexation, proper selection of co-solutes and solution conditions plays a crucial role. The onset of (bio)macromolecular complexation occurs even on the so-called "wrong side" of the protein isoionic point where both the protein and the polyelectrolyte are net like-charged. To gain mechanistic insights into the modulatory role of salts (NaCl, NaBr, and NaI) and sugars (sucrose and sucralose) in protein-polyelectrolyte complexation under such conditions, interaction between bovine serum albumin (BSA) and sodium polystyrene sulfonate (NaPSS) at pH = 8.0 was studied by a combination of isothermal titration calorimetry, fluorescence spectroscopy, circular dichroism, and thermodynamic modeling. The BSA-NaPSS complexation proceeds by two binding processes (first, formation of intrapolymer complexes and then formation of interpolymer complexes), both driven by favorable electrostatic interactions between the negatively charged sulfonic groups (-SO3-) of NaPSS and positively charged patches on the BSA surface. Two such positive patches were identified, each responsible for one of the two binding processes. The presence of salts screened both short-range attractive and long-range repulsive electrostatic interactions between both macromolecules, resulting in a nonmonotonic dependence of the binding affinity on the total ionic strength for both binding processes. In addition, distinct anion-specific effects were observed (NaCl < NaBr < NaI). The effect of sugars was less pronounced: sucrose had no effect on the complexation, but its chlorinated analogue, sucralose, promoted it slightly due to the screening of long-range repulsive electrostatic interactions between BSA and NaPSS. Although short-range non-electrostatic interactions are frequently mentioned in the literature in relation to BSA or NaPSS, we found that the main driving force of complexation on the "wrong side" are electrostatic interactions.

A new fibrillization mechanism of ß-lactoglobulin in glycine solutions.

Even though amyloid aggregates were discovered many years ago the mechanism of their formation is still a mystery. Because of their connection to many of untreatable neurodegenerative diseases the motivation for finding a common aggregation path is high. We report a new high heat induced fibrillization path of a model protein ß-lactoglobulin (BLG) when incubated in glycine instead of water at pH 2. By combining atomic force microscopy (AFM), transmission emission microscopy (TEM), dynamic light scattering (DLS) and circular dichroism (CD) we predict that the basic building blocks of fibrils made in glycine are not peptides, but rather spheroid oligomers of different height that form by stacking of ring-like structures. Spheroid oligomers linearly align to form fibrils by opening up and combining. We suspect that glycine acts as an hydrolysation inhibitor which consequently promotes a different fibrillization path. By combining the known data on fibrillization in water with our experimental conclusions we come up with a new fibrillization scheme for BLG. We show that by changing the fibrillization conditions just by small changes in buffer composition can dramatically change the aggregation pathway and the effect of buffer shouldn't be neglected. Fibrils seen in our study are also gaining more and more attention because of their pore-like structure and a possible cytotoxic mechanism by forming pernicious ion-channels. By preparing them in a simple model system as BLG we opened a new way to study their formation.

Phosphorylated and Phosphomimicking Variants May Differ-A Case Study of 14-3-3 Protein.

Protein phosphorylation is a critical mechanism that biology uses to govern cellular processes. To study the impact of phosphorylation on protein properties, a fully and specifically phosphorylated sample is required although not always achievable. Commonly, this issue is overcome by installing phosphomimicking mutations at the desired site of phosphorylation. 14-3-3 proteins are regulatory protein hubs that interact with hundreds of phosphorylated proteins and modulate their structure and activity. 14-3-3 protein function relies on its dimeric nature, which is controlled by Ser58 phosphorylation. However, incomplete Ser58 phosphorylation has obstructed the detailed study of its effect so far. In the present study, we describe the full and specific phosphorylation of 14-3-3ζ protein at Ser58 and we compare its characteristics with phosphomimicking mutants that have been used in the past (S58E/D). Our results show that in case of the 14-3-3 proteins, phosphomimicking mutations are not a sufficient replacement for phosphorylation. At physiological concentrations of 14-3-3ζ protein, the dimer-monomer equilibrium of phosphorylated protein is much more shifted towards monomers than that of the phosphomimicking mutants. The oligomeric state also influences protein properties such as thermodynamic stability and hydrophobicity. Moreover, phosphorylation changes the localization of 14-3-3ζ in HeLa and U251 human cancer cells. In summary, our study highlights that phosphomimicking mutations may not faithfully represent the effects of phosphorylation on the protein structure and function and that their use should be justified by comparing to the genuinely phosphorylated counterpart.

Quantitation of Human 14-3-3ζ Dimerization and the Effect of Phosphorylation on Dimer-monomer Equilibria.

14-3-3 proteins are universal regulatory proteins and their function depends on their oligomeric form which may alter between the monomeric, homodimeric and heterodimeric states. The populations of individual oligomeric forms are controlled by Kd values of the dimer-monomer equilibria between the involved isoforms. This complex picture is extended by post-translational modifications, e.g. phosphorylation. In this work, we describe the equilibria between monomers, homo- and heterodimers of the 14-3-3ζ isoform in the unmodified and phosphorylated form. To cover a wide range of dimerization affinities, we combined solution NMR, microscale thermophoresis, native PAGE, and a set of novel fluorescence assays. Using a FRET based assay, we also determined the kinetic parameters of dimerization. We found that phosphorylation of 14-3-3ζ at Ser58 increases its homodimeric Kd value by 6 orders of magnitude. The presented assays allow to efficiently monitor 14-3-3ζ dimerization as a function of external factors, such as temperature, salt concentration, and client protein binding. For instance, we obtained values of both transient and equilibrium thermodynamic constants for the dimerization, and observed a substantial decrease of 14-3-3ζ dimer dissociation rate upon binding to the doubly phosphorylated regulatory domain of tyrosine hydroxylase. In summary, our work provides a conceptual framework to characterise the isoform exchanges of homo- and heterodimers which can significantly deepen our knowledge about the regulatory function of 14-3-3 proteins.

NMR Studies of Tau Protein in Tauopathies.

Tauopathies, including Alzheimer's disease (AD), are the most troublesome of all age-related chronic conditions, as there are no well-established disease-modifying therapies for their prevention and treatment. Spatio-temporal distribution of tau protein pathology correlates with cognitive decline and severity of the disease, therefore, tau protein has become an appealing target for therapy. Current knowledge of the pathological effects and significance of specific species in the tau aggregation pathway is incomplete although more and more structural and mechanistic insights are being gained using biophysical techniques. Here, we review the application of NMR to structural studies of various tau forms that appear in its aggregation process, focusing on results obtained from solid-state NMR. Furthermore, we discuss implications from these studies and their prospective contribution to the development of new tauopathy therapies.

Universal two-point interaction of mediator KIX with 9aaTAD activation domains.

The nine-amino-acid activation domain (9aaTAD) is defined by a short amino acid pattern including two hydrophobic regions (positions p3-4 and p6-7). The KIX domain of mediator transcription CBP interacts with the 9aaTAD domains of transcription factors MLL, E2A, NF-kB, and p53. In this study, we analyzed the 9aaTADs-KIX interactions by nuclear magnetic resonance. The positions of three KIX helixes α1-α2-α3 are influenced by sterically-associated hydrophobic I611, L628, and I660 residues that are exposed to solvent. The positions of two rigid KIX helixes α1 and α2 generate conditions for structural folding in the flexible KIX-L12-G2 regions localized between them. The three KIX I611, L628, and I660 residues interact with two 9aaTAD hydrophobic residues in positions p3 and p4 and together build a hydrophobic core of five residues (5R). Numerous residues in 9aaTAD position p3 and p4 could provide this interaction. Following binding of the 9aaTAD to KIX, the hydrophobic I611, L628, and I660 residues are no longer exposed to solvent and their position changes inside the hydrophobic core together with position of KIX α1-α2-α3 helixes. The new positions of the KIX helixes α1 and α2 allow the KIX-L12-G2 enhanced formation. The second hydrophobic region of the 9aaTAD (positions p6 and p7) provides strong binding with the KIX-L12-G2 region. Similarly, multiple residues in 9aaTAD position p6 and p7 could provide this interaction. In conclusion, both 9aaTAD regions p3, p4 and p6, p7 provide co-operative and highly universal binding to mediator KIX. The hydrophobic core 5R formation allows new positions of the rigid KIX α-helixes and enables the enhanced formation of the KIX-L12-G2 region. This contributes to free energy and is the key for the KIX-9aaTAD binding. Therefore, the 9aaTAD-KIX interactions do not operate under the rigid key-and-lock mechanism what explains the 9aaTAD natural variability.