The propensity of the unstructured protein α-synuclein to undergo a conformational transition to fibrillar aggregates was harnessed to prepare an upconverting organic-inorganic hybrid film composed of Au nanoparticles (NPs) and Yb3+, Er3+-activated CaF2 NPs, with α-synuclein serving as a structural linker. The 2D nanomaterial appears as a quasi-monolayer film, composed of distributed AuNPs and tightly packed CaF2:Yb,Er NPs. The film shows excellent upconversion emission arising from the Er3+ ions following excitation of the Yb3+ ions under 980 nm laser radiation. Optical thermometry investigations were conducted in various media (air, H2O, and D2O) to assess the performance of the film as an optical nanothermometer. The thermometric calibration curves obtained within the physiological temperature range (25-60°C) support its potential application in 2D optical nanothermometry. By evaluating the energy gap between the two thermalized levels and calibrating the upconversion at one temperature, the nanocomposite film was found to perform reliably as a primary thermometer. Furthermore, image-based optical thermometry has demonstrated that upconversion emission can be successfully employed for local temperature estimation, thereby enabling image-based 2D optical thermometry with micrometer-scale spatial resolution.

Abnormal accumulation of tau fibrillar aggregates is a hallmark of tauopathies, including Alzheimer's disease. Targeting tau aggregation represents a promising strategy for preventing and treating neurological disorders, especially using natural compounds with favorable safety profiles. In this study, we investigated a hydroalcoholic extract of Cinnamomum cassia buds (BCHE) and its major components, cinnamaldehyde and shikimic acid, for their effects in modulating tau repeat domain aggregation and liquid-liquid phase separation. In vitro results show that BCHE and cinnamaldehyde inhibit tau aggregate maturation, promoting the formation of nonfibrillar, off-pathway species and modulating condensate formation. These alternative aggregates exhibit reduced cytotoxicity in SH-SY5Y neuroblastoma cells and lower seeding capacity than canonical fibrils. BCHE also contains compounds capable of binding preformed tau fibrils. Overall, these findings suggest a novel mechanism by which cinnamon-derived bioactive molecules mitigate tau aggregation and reduce its cellular toxicity, highlighting their potential as neuroprotective agents.

Alpha-synuclein (αS) is a neuronal protein implicated in synaptic regulation, whose pathological aggregation is a hallmark of synucleinopathies, including Parkinson's disease. Despite its physiological importance, its intrinsically disordered nature and conformational plasticity complicate both mechanistic understanding and therapeutic targeting. Here, we investigate the use of protein-functionalized nanoparticles (NPs) as tools to modulate and probe αS assembly pathways, including aggregation and liquid-liquid phase separation. We designed three nanoconjugates by covalently attaching single-cysteine αS variants at positions 18, 76, and 140, spanning the protein's three functional regions, onto PEG-coated silica NPs. These conjugates preserved the disordered character of αS, exhibited low cytotoxicity, and were internalized into neuronal cells. Functional assays revealed site-dependent effects on aggregation: NP-αSA76C significantly delayed αS fibril formation and further inhibited aggregation of the amyloidogenic tau protein, suggesting interference with nucleation mechanisms, while NP-αSA18C modestly accelerated aggregation and showed strong affinity for mature fibrils. All nanoconjugates partitioned into αS condensates, though without marked site-specific differences, and interacted with model lipid membranes. Overall, αS-functionalized NPs influence protein aggregation and condensate interactions, with context-dependent site specificity. These findings highlight the importance of conjugation position in tuning NP-protein interactions and underscore the potential of these nanoconjugates for investigating and modulating pathological protein assembly in neurodegenerative diseases.

Posttranslational modifications can critically affect conformational changes of amyloid-forming proteins. Ubiquitination of the microtubule-associated tau protein, an intrinsically disordered biomolecule, has been proposed to influence the formation of filamentous deposits in neurodegenerative conditions. Given the reported link between aggregation propensity and intrinsic structural preferences (e.g., transient extended structural motifs or tertiary contacts) in disordered proteins, we sought to explore the conformational landscape of ubiquitinated tau. Exploiting selective conjugation reactions, we produced single- and double-monoubiquitinated protein samples. Next, we examined the ubiquitinated species from different standpoints using NMR spectroscopy, small-angle X-ray scattering experiments, and native ion mobility-mass spectrometry (IM-MS). Moreover, we obtained atomistic representations of the conformational ensembles via scaled MD calculations, consistent with the experimental data. Modifying the repeat domain of tau with ubiquitin had a limited effect on secondary structure propensities and local mobility of distal regions. Instead, ubiquitination enhanced the compaction of the conformational ensemble, with the effect modulated by the site and the number of modifications. Native IM-MS patterns pinpointed similarities and differences between distinct tau proteoforms. It emerges that ubiquitination exerts a position-specific influence on the conformational distribution of tau molecules. This study reveals the unique conformational features of ubiquitinated forms of tau and points to their potential impact on aggregation and phase separation propensities, offering clues for a better understanding of disease-related structural alterations.

The ubiquitin proteasome system is a critical regulator of proteostasis and shows altered activity and composition in neurodegenerative diseases affecting both the brain (e.g., Alzheimer's disease) and the retina/optic nerve (e.g., age-related macular degeneration, glaucoma). A common feature of neurodegeneration is the progressive accumulation of amyloidogenic proteins such as beta-amyloid and tau protein (MAPT gene). There is compelling evidence that the aggregation propensity of tau protein is regulated by post-synthetic modifications including phosphorylation and ubiquitylation. These alterations are gaining increasing pathological relevance not only for brain tauopathies but also for the retinal/optic nerve degenerative diseases. In this regard, site-specific mono-ubiquitylated (Ub) tau proteoforms, have been recently identified in neurodegenerative brains. In this work, the cleavage patterns of the uncapped 20S proteasome acting on mono-Ub regio-isomers of tauK18, which covers the 4RD domain, have been unveiled by using SpectraSage, a novel proteomics software conceived for the MS1 identification of complex branched peptide and here introduced for the first time. Ub position was found to affect regio-isomers susceptibility to proteolysis and unexpectedly long Ub-tauK18 branched peptides have been identified, proving distinct catalytic preferences. These findings show that the 20S digests mono-Ub proteins through specific enzymatic mechanisms and the implications of the latter on neurodegeneration are discussed.

Greater concentrations of hydrosulfide lead to the prebiotic formation of higher nuclearity Fe–S peptides, culminating in a putative nitrogenase-like [6Fe–9S] cluster. Higher nuclearity clusters are more stable with lower reduction potential.

The interaction between the microtubule associated protein Tau and the tyrosine kinase Fyn is believed to play a pivotal role in the early stage of Alzheimer's disease. Previous studies have identified the SRC Homology 3 (SH3) domain of Fyn as the binding receptor of several proline-rich motifs in Tau. However, the role of each proline-rich motif and their interplay in molecular recognition are still unclear. In this work, we investigated the mechanism of Fyn-SH3 recognition by the multiple PxxP sites inserted within the full-length Tau protein by using nuclear magnetic resonance (NMR) spectroscopy combined with computational, calorimetric and in-cell FRET (Förster resonance energy transfer) methods. Both in vitro and in-cell experiments revealed no single binding site strictly necessary for the binding. Instead, Fyn-SH3 contacts full-length Tau on multiple hot spot regions, located over a distance of 85 residues, through global moderate-to-low affinity interactions. Beyond two principal regions containing classical PxxP motifs, we identified a novel non-canonical binding site at the beginning of the microtubule binding domain. Our study indicates that multiple binding sites in Tau are involved in the interaction, making Tau an adaptable recognition surface that can function when single consensus motifs are deleted.

Nuclear factor κB (NF-κB) signaling is a central pathway regulating a plethora of cellular functions. Here, we find that RNF32, a RING E3 ubiquitin ligase whose expression is enriched in murine intestinal stem cells, regulates the activity of the IκB kinase (IKK) complex, the signal integration hub for NF-κB activation. The E3 ligase activity of RNF32 depends on calmodulin, the primary calcium sensor in eukaryotic cells. Increased levels of intracellular calcium ion (Ca2+) induce RNF32 binding to calmodulin, RNF32 activation, and autoubiquitylation. In turn, polyubiquitin chains conjugated to RNF32 recruit NEMO, the regulatory subunit of the IKK complex. Moreover, Ca2+ rise triggers RNF32 phase separation, which is required for the formation of NEMO condensates and IKK activation. Finally, we show that RNF32 is required for NF-κB activation triggered by bacterial lipopolysaccharides. Collectively, our findings uncover a mechanism controlling NF-κB signaling in the intestinal epithelium.

The microtubule-associated protein tau is an intrinsically disordered protein highly expressed in neuronal axons. In healthy neurons, tau regulates microtubule dynamics and neurite outgrowth. However, pathological conditions can trigger aberrant tau aggregation into insoluble filaments, a hallmark of neurodegenerative disorders known as tauopathies. Tau undergoes diverse posttranslational modifications (PTMs), suggesting complex regulation and potentially varied functions. Among PTMs, the role and mechanisms of ubiquitination in physiology and disease have remained enigmatic. The past three decades have witnessed the emergence of key studies on tau protein ubiquitination. In this concept, we discuss how these investigations have begun to shed light on the ubiquitination patterns of physiological and pathological tau, the responsible enzymatic machinery, and the influence of ubiquitination on tau aggregation. We also provide an overview of the semi-synthetic methods that have enabled in vitro investigations of conformational transitions of tau induced by ubiquitin modification. Finally, we discuss future perspectives in the field necessary to elucidate the molecular mechanisms of tau ubiquitination and clearance.

Protein semisynthesis approaches are key for gaining insights into the effects of post-translational modifications (PTMs) on the structure and function of modified proteins. Among PTMs, ubiquitination involves the conjugation of a small protein modifier to a substrate amino acid residue and is unique in controlling a variety of cellular processes. Interest has grown in understanding the role of ubiquitination in neurodegenerative conditions, including tauopathies. The latter are characterized by the accumulation of the intrinsically disordered protein tau in the form of neurofibrillary tangles in the brains of patients. The presence of ubiquitinated tau in the pathological aggregates suggests that ubiquitination might play a role in the formation of abnormal protein deposits. In this study, we developed a new strategy, based on dehydroalanine chemistry, to install wild type ubiquitin on a tau repeat domain construct with site-specificity. We optimized a three-step reaction which yielded a good amount of highly pure tau repeat domain ubiquitinated in position 353. The structural features of the conjugate were examined by circular dichroism and NMR spectroscopy. The ubiquitinated tau was challenged in a number of assays: fibrils formation under aggregating conditions in vitro, chemical stability upon exposure to a variety of biological media including cell extracts, and internalization into astrocytes. The results demonstrated the wide applicability of the new semisynthetic strategy for the investigation of ubiquitinated substrates in vitro or in cell, and in particular for studying if ubiquitination has a role in the molecular mechanisms that underlie the aberrant transition of tau into pathological aggregates.

We have applied a proteolysis targeting chimera (PROTAC) technology to obtain a peptidomimetic molecule able to trigger the degradation of SARS-CoV-2 3-chymotrypsin-like protease (3CL(Pro)). The PROTAC molecule was designed by conjugating a GC-376 based dipeptidyl 3CL(Pro) ligand to a pomalidomide moiety through a piperazine-piperidine linker. NMR and crystallographic data complemented with enzymatic and cellular studies showed that (i) the dipeptidyl moiety of PROTAC binds to the active site of the dimeric state of SARS-CoV-2 3CL(Pro) forming a reversible covalent bond with the sulfur atom of catalytic Cys145, (ii) the linker and the pomalidomide cereblon-ligand of PROTAC protrude from the protein, displaying a high degree of flexibility and no interactions with other regions of the protein, and (iii) PROTAC reduces the protein levels of SARS-CoV-2 3CL(Pro) in cultured cells. This study paves the way for the future applicability of peptidomimetic PROTACs to tackle 3CL(Pro)-dependent viral infections.

In Alzheimer's disease and related disorders called tauopathies, the microtubule-associated protein tau accumulates in the brain in the form of amyloid-like supramolecular filaments. As an intrinsically disordered protein, tau undergoes many post-translational modifications, including ubiquitination. Alterations to the levels of ubiquitination of tau have been observed at various stages of neurodegenerative conditions. We focus on proteoform-specific interrogations to obtain mechanistic insight into the effects of ubiquitination on disease-related conformational transitions of tau. Single and double ubiquitination of tau at residues Lys311 and Lys317 is strongly associated with pathological conditions. In this study, we leveraged disulfide-directed chemistry to install ubiquitin at one or both of those positions in the isolated microtubule-binding repeat domain of tau. We obtained homogeneously modified tau proteins and observed that they retained disordered character in solution. We found that ubiquitination in position 317 (with or without ubiquitination in position 311) impaired the formation of ordered fibrillar structures via oligomeric intermediates. Since the transition to fibrillar species may proceed via an alternative condensation pathway involving liquid droplet intermediates, we further tested the ability of the ubiquitinated proteoforms to phase separate. Single monoubiquitinated tau species were able to coacervate, however no liquid droplets were observed for the double ubiquitinated form. Taken together, the data indicate that double ubiquitination in the third repeat of tau disfavors the formation of amyloid aggregates by distinct mechanisms, suggesting that the presence of ubiquitinated residues 311 and 317 in insoluble tau may result from modifications in advanced stages of aggregation. These findings contribute to our understanding of the influence of site-specific ubiquitination on the pathological conformational transitions of a prototypical intrinsically disordered protein.

Espresso coffee is among the most consumed beverages in the world. Recent studies report a protective activity of the coffee beverage against neurodegenerative disorders such as Alzheimer's disease. Alzheimer's disease belongs to a group of disorders, called tauopathies, which are characterized by the intraneuronal accumulation of the microtubule-associated protein tau in fibrillar aggregates. In this work, we characterized by NMR the molecular composition of the espresso coffee extract and identified its main components. We then demonstrated with in vitro and in cell experiments that the whole coffee extract, caffeine, and genistein have biological properties in preventing aggregation, condensation, and seeding activity of the repeat region of tau. We also identified a set of coffee compounds capable of binding to preformed tau fibrils. These results add insights into the neuroprotective potential of espresso coffee and suggest candidate molecular scaffolds for designing therapies targeting monomeric or fibrillized forms of tau.

Post-translational modifications of Tau are emerging as key players in determining the onset and progression of different tauopathies such as Alzheimer's disease, and are recognized to mediate the structural diversity of the disease-specific Tau amyloids. Here we show that the E3 ligase CHIP catalyzes the site-specific ubiquitination of Tau filaments both in vitro and in cellular models, proving that also Tau amyloid aggregates are direct substrate of PTMs. Transmission electron microscopy and mass spectrometry analysis on ubiquitin-modified Tau amyloids revealed that the conformation of the filaments restricts CHIP-mediated ubiquitination to specific positions of the repeat domain, while only minor alterations in the structure of the fibril core were inferred using seeding experiments in vitro and in a cell-based tauopathy model. Overexpression of CHIP significantly increased the ubiquitination of exogenous PHF, proving that the ligase can interact and modify Tau aggregates also in a complex cellular environment.

Liquid-liquid phase separation (LLPS) of biopolymers to form condensates is a widespread phenomenon in living cells. Agents that target or alter condensation can help uncover elusive physiological and pathological mechanisms. Owing to their unique material properties and modes of interaction with biomolecules, nanoparticles represent attractive condensate-targeting agents. Our work focused on elucidating the interaction between ultrasmall gold nanoparticles (usGNPs) and diverse types of condensates of tau, a representative phase-separating protein associated with neurodegenerative disorders. usGNPs attract considerable interest in the biomedical community due to unique features, including emergent optical properties and good cell penetration. We explored the interaction of usGNPs with reconstituted self-condensates of tau, two-component tau/polyanion and three-component tau/RNA/alpha-synuclein coacervates. The usGNPs were found to concentrate into condensed liquid droplets, consistent with the formation of dynamic client (nanoparticle) - scaffold (tau) interactions, and were observable thanks to their intrinsic luminescence. Furthermore, usGNPs were capable to promote LLPS of a protein domain which is unable to phase separate on its own. Our study demonstrates the ability of usGNPs to interact with and illuminate protein condensates. We anticipate that nanoparticles will have broad applicability as nanotracers to interrogate phase separation, and as nanoactuators controlling the formation and dissolution of condensates.

Understanding the interactions between nanoparticles (NPs) and proteins is crucial for the successful application of NPs in biological contexts. Protein adsorption is dependent on particle size, and protein binding to ultrasmall (1-3 nm) NPs is considered to be generally weak. However, most studies have involved structured biomacromolecules, while the interactions of ultrasmall NPs with intrinsically disordered proteins (IDPs) have remained elusive. IDPs are abundant in eukaryotes and found to associate with NPs intracellularly. As a model system, we focused on ultrasmall gold nanoparticles (usGNPs) and tau, a cytosolic IDP associated with Alzheimer's disease. Using site-resolved NMR, steady-state fluorescence, calorimetry, and circular dichroism, we reveal that tau and usGNPs form stable multimolecular assemblies, representing a new type of nano-bio interaction. Specifically, the observed interaction hot spots explain the influence of usGNPs on tau conformational transitions, with implications for the intracellular targeting of aberrant IDP aggregation.

The multi-site ubiquitination of Tau protein found in Alzheimer's disease filaments hints at the failed attempt of neurons to remove early toxic species. The ubiquitin-dependent degradation of Tau is regulated in vivo by the E3 ligase CHIP, a quality controller of the cell proteome dedicated to target misfolded proteins for degradation. In our study, by using site-resolved NMR, biochemical and computational methods, we elucidate the structural determinants underlying the molecular recognition between the ligase and its intrinsically disordered substrate. We reveal a multi-domain dynamic interaction that explains how CHIP can direct ubiquitination of Tau at multiple sites even in the absence of chaperones, including its typical partner Hsp70/Hsc70. Our findings thus provide mechanistic insight into the chaperone-independent engagement of a disordered protein by its E3 ligase.

Intrinsically disordered proteins (IDPs) are increasingly found to be associated with irreversible neurodegenerative disorders. The protein tau is a prototypical IDP whose abnormal aggregation into insoluble filaments is a major hallmark of Alzheimer's disease. The view has emerged that aggregation may proceed via alternative pathways involving oligomeric intermediates or phase-separated liquid droplets. Nanoparticles (NPs) offer significant potential for probing the mechanisms of protein fibrillation and may be capable of redirecting conformational transitions. Here, we camouflaged dye-doped silica NPs through functionalization with tau molecules to impart them the ability to associate with protein assemblies such as aggregates or condensates. The prepared NP-tau conjugates showed little influence on the aggregation kinetics and morphology of filamentous aggregates of tau but were found to associate with the filaments. Moreover, NP-tau conjugates were recruited and concentrated into polyanion-induced condensates of tau, driven by multivalent electrostatic interactions, thereby illuminating liquid droplets and their time-dependent transformation, as observed by fluorescence microscopy. NP-tau conjugates were capable of entering human neuroglioma cells and were not cytotoxic. Hence, we propose that NP-tau conjugates could serve as nanotracers for in vitro and in-cell studies to target and visualize tau assemblies and condensates, contributing to an explanation for the molecular mechanisms of abnormal protein aggregation.