Phosphorylation of the overlooked tyrosine 310 regulates the structure, aggregation, and microtubule- and lipid-binding properties of Tau.

The microtubule-associated protein Tau is implicated in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease. Increasing evidence suggests that post-translational modifications play critical roles in regulating Tau's normal functions and its pathogenic properties in tauopathies. Very little is known about how phosphorylation of tyrosine residues influences the structure, aggregation, and microtubule- and lipid-binding properties of Tau. Here, we sought to determine the relative contributions of phosphorylation of one or several of the five tyrosine residues in Tau (Tyr-18, -29, -197, -310, and -394) to the regulation of its biophysical, aggregation, and functional properties. We used a combination of site-specific mutagenesis and in vitro phosphorylation by c-Abl kinase to generate Tau species phosphorylated at all five tyrosine residues, all tyrosine residues except Tyr-310 or Tyr-394 (pTau-Y310F and pTau-Y394F, respectively) and Tau phosphorylated only at Tyr-310 or Tyr-394 (4F/pTyr-310 or 4F/pTyr-394). We observed that phosphorylation of all five tyrosine residues, multiple N-terminal tyrosine residues (Tyr-18, -29, and -197), or specific phosphorylation only at residue Tyr-310 abolishes Tau aggregation and inhibits its microtubule- and lipid-binding properties. NMR experiments indicated that these effects are mediated by a local decrease in ß-sheet propensity of Tau's PHF6 domain. Our findings underscore Tyr-310 phosphorylation has a unique role in the regulation of Tau aggregation, microtubule, and lipid interactions. These results also highlight the importance of conducting further studies to elucidate the role of Tyr-310 in the regulation of Tau's normal functions and pathogenic properties.

Unraveling the complexity of amyloid polymorphism using gold nanoparticles and cryo-EM.

Increasing evidence suggests that amyloid polymorphism gives rise to different strains of amyloids with distinct toxicities and pathology-spreading properties. Validating this hypothesis is challenging due to a lack of tools and methods that allow for the direct characterization of amyloid polymorphism in hydrated and complex biological samples. Here, we report on the development of 11-mercapto-1-undecanesulfonate-coated gold nanoparticles (NPs) that efficiently label the edges of synthetic, recombinant, and native amyloid fibrils derived from different amyloidogenic proteins. We demonstrate that these NPs represent powerful tools for assessing amyloid morphological polymorphism, using cryogenic transmission electron microscopy (cryo-EM). The NPs allowed for the visualization of morphological features that are not directly observed using standard imaging techniques, including transmission electron microscopy with use of the negative stain or cryo-EM imaging. The use of these NPs to label native paired helical filaments (PHFs) from the postmortem brain of a patient with Alzheimer's disease, as well as amyloid fibrils extracted from the heart tissue of a patient suffering from systemic amyloid light-chain amyloidosis, revealed a high degree of homogeneity across the fibrils derived from human tissue in comparison with fibrils aggregated in vitro. These findings are consistent with, and strongly support, the emerging view that the physiologic milieu is a key determinant of amyloid fibril strains. Together, these advances should not only facilitate the profiling and characterization of amyloids for structural studies by cryo-EM, but also pave the way to elucidate the structural basis of amyloid strains and toxicity, and possibly the correlation between the pathological and clinical heterogeneity of amyloid diseases.

Resolving molecule-specific information in dynamic lipid membrane processes with multi-resonant infrared metasurfaces.

A multitude of biological processes are enabled by complex interactions between lipid membranes and proteins. To understand such dynamic processes, it is crucial to differentiate the constituent biomolecular species and track their individual time evolution without invasive labels. Here, we present a label-free mid-infrared biosensor capable of distinguishing multiple analytes in heterogeneous biological samples with high sensitivity. Our technology leverages a multi-resonant metasurface to simultaneously enhance the different vibrational fingerprints of multiple biomolecules. By providing up to 1000-fold near-field intensity enhancement over both amide and methylene bands, our sensor resolves the interactions of lipid membranes with different polypeptides in real time. Significantly, we demonstrate that our label-free chemically specific sensor can analyze peptide-induced neurotransmitter cargo release from synaptic vesicle mimics. Our sensor opens up exciting possibilities for gaining new insights into biological processes such as signaling or transport in basic research as well as provides a valuable toolkit for bioanalytical and pharmaceutical applications.

Discovery and characterization of stable and toxic Tau/phospholipid oligomeric complexes.

The microtubule-associated protein Tau plays a central role in the pathogenesis of Alzheimer's disease. Although Tau interaction with membranes is thought to affect some of its physiological functions and its aggregation properties, the sequence determinants and the structural and functional consequences of such interactions remain poorly understood. Here, we report that the interaction of Tau with vesicles results in the formation of highly stable protein/phospholipid complexes. These complexes are toxic to primary hippocampal cultures and are detected by MC-1, an antibody recognizing pathological Tau conformations. The core of these complexes is comprised of the PHF6* and PHF6 hexapeptide motifs, the latter in a ß-strand conformation. Studies using Tau-derived peptides enabled the design of mutants that disrupt Tau interactions with phospholipids without interfering with its ability to form fibrils, thus providing powerful tools for uncoupling these processes and investigating the role of membrane interactions in regulating Tau function, aggregation and toxicity.

Microtubule-Binding R3 Fragment from Tau Self-Assembles into Giant Multistranded Amyloid Ribbons.

Tau protein and its fragments self-assemble into amyloid fibrils in the presence of polyanions, such as heparin. By combining microscopy, scattering, and spectroscopy techniques, we studied the aggregation of the 26-mer Tau-derived peptide alone, Tau(306-327), the third repeat fragment (R3) of the microtubule-binding domain. We show that: i) the sole Tau(306-327) can self-assemble into amyloid fibrils without the need of aggregation-promoting polyanions; ii) the resulting structures consist of surprisingly large, well-ordered 2D laminated flat ribbons, with a log-normal distribution of the lateral width, reaching the unprecedented lateral size of 350 nm and/or 45 individual protofilaments, that is, the largest amyloid laminated structures ever observed for Tau or any other amyloidogenic sequence. Our results provide insight into the molecular determinants of Tau aggregation and open new perspectives in the understanding of the assembly of amyloid fibrils and ß-sheet-based biomaterials.

Elucidating the Role of Site-Specific Nitration of α-Synuclein in the Pathogenesis of Parkinson's Disease via Protein Semisynthesis and Mutagenesis.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and the presence of intraneuronal inclusions consisting of aggregated and post-translationally modified α-synuclein (α-syn). Despite advances in the chemical synthesis of α-syn and other proteins, the generation of site-specifically nitrated synthetic proteins has not been reported. Consequently, it has not been possible to determine the roles of nitration at specific residues in regulating the physiological and pathogenic properties of α-syn. Here we report, for the first time, the site-specific incorporation of 3-nitrotyrosine at different regions of α-syn using native chemical ligation combined with a novel desulfurization strategy. This strategy enabled us to investigate the role of nitration at single or multiple tyrosine residues in regulating α-syn structure, membrane binding, oligomerization, and fibrils formation. We demonstrate that different site-specifically nitrated α-syn species exhibit distinct structural and aggregation properties and exhibit reduced affinity to negatively charged vesicle membranes. We provide evidence that intermolecular interactions between the N- and C-terminal regions of α-syn play critical roles in mediating nitration-induced α-syn oligomerization. For example, when Y39 is not available for nitration (Y39F and Y39/125F), the extent of cross-linking is limited mostly to dimer formation, whereas mutants in which Y39 along with one or multiple C-terminal tyrosines (Y125F, Y133F, Y136F and Y133/136F) can still undergo nitration readily to form higher-order oligomers. Our semisynthetic strategy for generating site-specifically nitrated proteins opens up new possibilities for investigating the role of nitration in regulating protein structure and function in health and disease.

The novel Parkinson's disease linked mutation G51D attenuates in vitro aggregation and membrane binding of α-synuclein, and enhances its secretion and nuclear localization in cells.

A novel mutation in the α-Synuclein (α-Syn) gene "G51D" was recently identified in two familial cases exhibiting features of Parkinson's disease (PD) and multiple system atrophy (MSA). In this study, we explored the impact of this novel mutation on the aggregation, cellular and biophysical properties of α-Syn, in an attempt to unravel how this mutant contributes to PD/MSA. Our results show that the G51D mutation significantly attenuates α-Syn aggregation in vitro. Moreover, it disrupts local helix formation in the presence of SDS, decreases binding to lipid vesicles C-terminal to the site of mutation and severely inhibits helical folding in the presence of acidic vesicles. When expressed in yeast, α-Syn(G51D) behaves similarly to α-Syn(A30P), as both exhibit impaired membrane association, form few inclusions and are non-toxic. In contrast, enhanced secreted and nuclear levels of the G51D mutant were observed in mammalian cells, as well as in primary neurons, where α-Syn(G51D) was enriched in the nuclear compartment, was hyper-phosphorylated at S129 and exacerbated α-Syn-induced mitochondrial fragmentation. Finally, post-mortem human brain tissues of α-Syn(G51D) cases were examined, and revealed only partial colocalization with nuclear membrane markers, probably due to post-mortem tissue delay and fixation. These findings suggest that the PD-linked mutations may cause neurodegeneration via different mechanisms, some of which may be independent of α-Syn aggregation.