RESUMO
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 (3CLPro). The PROTAC molecule was designed by conjugating a GC-376 based dipeptidyl 3CLPro 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 3CLPro 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 3CLPro in cultured cells. This study paves the way for the future applicability of peptidomimetic PROTACs to tackle 3CLPro-dependent viral infections.
RESUMO
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.
Assuntos
Doença de Alzheimer , Tauopatias , Humanos , Proteínas tau/metabolismo , Doença de Alzheimer/prevenção & controle , Doença de Alzheimer/metabolismo , Tauopatias/prevenção & controle , Tauopatias/metabolismo , Cafeína/farmacologia , Extratos VegetaisRESUMO
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.
Assuntos
Condensados Biomoleculares , Nanopartículas Metálicas , Humanos , Ouro , Luminescência , Domínios ProteicosRESUMO
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.
Assuntos
Doença de Alzheimer , Proteínas Intrinsicamente Desordenadas , Humanos , Proteínas tau/metabolismo , Proteínas Amiloidogênicas , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Ubiquitinação , Ubiquitina/metabolismoRESUMO
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.
Assuntos
Proteínas Intrinsicamente Desordenadas , Nanopartículas Metálicas , Ouro/química , Proteínas Intrinsicamente Desordenadas/química , Ligação ProteicaRESUMO
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.
Assuntos
Doença de Alzheimer , Nanopartículas , Doença de Alzheimer/metabolismo , Proteínas Amiloidogênicas , Humanos , Agregados Proteicos , Conformação Proteica , Dióxido de Silício , Proteínas tauRESUMO
Calmodulin (CaM), a ubiquitous and highly conserved Ca2+-sensor protein involved in the regulation of over 300 molecular targets, has been recently associated with severe forms of lethal arrhythmia. Here, we investigated how arrhythmia-associated mutations in CaM localized at the C-terminal lobe alter the molecular recognition with Ryanodine receptor 2 (RyR2), specifically expressed in cardiomyocytes. We performed an extensive structural, thermodynamic, and kinetic characterization of the variants D95V/H in the EF3 Ca2+-binding motif and of the D129V and D131H/E variants in the EF4 motif, and probed their interaction with RyR2. Our results show that the specific structural changes observed for individual CaM variants do not extend to the complex with the RyR2 target. Indeed, some common alterations emerge at the protein-protein interaction level, suggesting the existence of general features shared by the arrhythmia-associated variants. All mutants showed a faster rate of dissociation from the target peptide than wild-type CaM. Integration of spectroscopic data with exhaustive molecular dynamics simulations suggests that, in the presence of Ca2+, functional recognition involves allosteric interactions initiated by the N-terminal lobe of CaM, which shows a lower affinity for Ca2+ compared to the C-terminal lobe in the isolated protein.
Assuntos
Arritmias Cardíacas , Calmodulina , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Arritmias Cardíacas/congênito , Arritmias Cardíacas/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Humanos , Ligação Proteica , Conformação Proteica , Domínios ProteicosRESUMO
The formation of biomolecular condensates has emerged as a crucial player both in neuronal physiology and neurodegeneration. Phase separation of the Alzheimer's related protein tau into liquid condensates is facilitated by polyanions and is regulated by post-translational modifications. Given the central role of ubiquitination in proteostasis regulation and signaling, we investigated the behavior of monoubiquitinated tau during formation of condensates. We ubiquitinated the lysine-rich, four-repeat domain of tau either unspecifically via enzymatic conjugation or in a position-specific manner by semisynthesis. Ubiquitin conjugation at specific sites weakened multivalent tau/RNA interactions and disfavored tau/heparin condensation. Yet, heterogeneous ubiquitination was tolerated during phase separation and stabilized droplets against aggregation-linked dissolution. Thus, we demonstrated that cofactor chemistry and site of modification affect the mesoscopic and molecular signatures of ubiquitinated tau condensates. Our findings suggest that ubiquitination could influence the physiological states and pathological transformations of tau in cellular condensates.
Assuntos
Doença de Alzheimer , Proteínas tau , Doença de Alzheimer/metabolismo , Humanos , Lisina/metabolismo , Ubiquitina/genética , Ubiquitinação , Proteínas tau/químicaRESUMO
BACKGROUND: The intrinsically disordered, amyloidogenic protein Tau associates with diverse classes of molecules, including proteins, nucleic acids, and lipids. Mounting evidence suggests that fatty acid molecules could play a role in the dysfunction of this protein, however, their interaction with Tau remains poorly characterized. METHODS: In a bid to elucidate the association of Tau with unsaturated fatty acids at the sub-molecular level, we carried out a variety of solution NMR experiments in combination with circular dichroism and fluorescence measurements. Our study shows that Tau4RD, the highly basic four-repeat domain of Tau, associates strongly with arachidonic and oleic acid assemblies in a high lipid/protein ratio, perturbing their supramolecular states and itself undergoing time-dependent structural adaptation. The structural signatures of Tau4RD/fatty acid aggregates appear similar for arachidonic acid and oleic acid, however, they are distinct from those of another prototypical intrinsically disordered protein, α-synuclein, when bound to these lipids, revealing protein-specific conformational adaptations. Both fatty acid molecules are found to invariably promote the self-aggregation of Tau4RD and of α-synuclein. CONCLUSIONS: This study describes the reciprocal influence that Tau4RD and fatty acids exert on their conformational states, contributing to our understanding of fundamental aspects of Tau/lipid co-assembly.
Assuntos
Ácido Araquidônico/farmacologia , Ácido Oleico/farmacologia , Proteínas tau/química , Proteínas tau/metabolismo , Dicroísmo Circular , Ácidos Graxos Insaturados/farmacologia , Humanos , Imageamento por Ressonância Magnética , Agregados Proteicos , Conformação Proteica , Domínios Proteicos , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMO
Ubiquitin, a protein modifier that regulates diverse essential cellular processes, is also a component of the protein inclusions characteristic of many neurodegenerative disorders. In Alzheimer's disease, the microtubule associated tau protein accumulates within damaged neurons in the form of cross-beta structured filaments. Both mono- and polyubiquitin were found linked to several lysine residues belonging to the region of tau protein that forms the structured core of the filaments. Thus, besides priming the substrate protein for proteasomal degradation, ubiquitin could also contribute to the assembly and stabilization of tau protein filaments. To advance our understanding of the impact of ubiquitination on tau protein aggregation and function, we applied disulfide-coupling chemistry to modify tau protein at position 353 with Lys48- or Lys63-linked di-ubiquitin, two representative polyubiquitin chains that differ in topology and structure. Aggregation kinetics experiments performed on these conjugates reveal that di-ubiquitination retards filament formation and perturbs the fibril elongation rate more than mono-ubiquitination. We further show that di-ubiquitination modulates tau-mediated microtubule assembly. The effects on tau protein aggregation and microtubule polymerization are essentially independent from polyubiquitin chain topology. Altogether, our findings provide novel insight into the consequences of ubiquitination on the functional activity and disease-related behavior of tau protein.
Assuntos
Ubiquitina/metabolismo , Proteínas tau/química , Proteínas tau/metabolismo , Dissulfetos/química , Humanos , Lisina/metabolismo , Agregados ProteicosRESUMO
The notion that nanoscale surfaces influence protein conformational transitions stimulates the investigation of fibrillogenic polypeptides adsorbing to nanomaterials. Alpha-synuclein (αS) is a prototypical amyloidogenic protein whose aggregation is associated with severe neurodegenerative disorders. We explored the interaction of αS with silica nanoparticles (SNPs) in diverse solution conditions, ranging from protein-free to protein-rich media. We found that the SNP-binding region of αS, determined by site-resolved NMR spectroscopy, was similar in simple buffer and blood serum. Competition binding experiments with isotopic homologues and different proteins showed that cosolutes elicited molecular exchange in a protein-specific manner. The interaction of an oxidized, fibrillation-resistant protein form with SNPs was similar to that of unmodified αS. SNPs, however, did not stimulate fibrillation of the oxidized protein, which remained fibrillation incompetent. CD experiments revealed SNP-induced perturbations of the structural properties of oxidized and non-oxidized αS. Thus, while αS binding to SNPs is essentially orthogonal to fibril formation, the interaction perturbs the distribution of conformational states populated by the protein in the colloidal suspension. This study sheds light on the dynamic nature of αS interactions with NPs, an aspect that crucially impacts on our ability to control aggregation of αS.
Assuntos
Nanopartículas/química , Agregação Patológica de Proteínas , Proteínas Recombinantes/química , Dióxido de Silício/química , alfa-Sinucleína/química , Humanos , Ligação Proteica , Conformação Proteica , Dobramento de ProteínaRESUMO
Liver fatty acid binding protein (L-FABP) is an abundant cytosolic protein playing a central role in intracellular lipid trafficking. The L-FABP T94A variant, originating from one of the most common polymorphisms in the FABP family, is associated with several lipid-related disorders. However, the molecular factors that determine the observed functional differences are currently unknown. In our work, we performed a high resolution comparative molecular analysis of L-FABP T94T and L-FABP T94A in their unbound states and in the presence of representative ligands of the fatty acid and bile acid classes. We collected residue-resolved NMR spectral fingerprints of the two variants, and compared secondary structures, backbone dynamics, side chain arrangements, binding site occupation, and intermolecular contacts. We found that threonine to alanine replacement did not result in strongly perturbed structural and dynamic features, although differences in oleic acid binding by the two variants were detected. Based on chemical shift perturbations at sites distant from position 94 and on differences in intermolecular contacts, we suggest that long-range communication networks in L-FABP propagate the effect of amino acid substitution at sites relevant for ligand binding or biomolecular recognition.