RESUMEN
Prion-like spread of disease-specific tau conformers is a hallmark of all tauopathies. A 19-residue probe peptide containing a P301L mutation and spanning the R2/R3 splice junction of tau folds and stacks into seeding-competent fibrils and induces aggregation of 4R, but not 3R tau. These tau peptide fibrils propagate aggregated intracellular tau over multiple generations, have a high ß-sheet content, a colocalized lipid signal, and adopt a well-defined U-shaped fold found in 4R tauopathy brain-derived fibrils. Fully atomistic replica exchange molecular dynamics (MD) simulations were used to compute the free energy landscapes of the conformational ensemble of the peptide monomers. These identified an aggregation-prohibiting ß-hairpin structure and an aggregation-competent U-fold unique to 4R tauopathy fibrils. Guided by MD simulations, we identified that the N-terminal-flanking residues to PHF6, which slightly vary between 4R and 3R isoforms, modulate seeding. Strikingly, when a single amino acid switch at position 305 replaced the serine of 4R tau with a lysine from the corresponding position in the first repeat of 3R tau, the seeding induced by the 19-residue peptide was markedly reduced. Conversely, a 4R tau mimic with three repeats, prepared by replacing those amino acids in the first repeat with those amino acids uniquely present in the second repeat, recovered aggregation when exposed to the 19-residue peptide. These peptide fibrils function as partial prions to recruit naive 4R tau-ten times the length of the peptide-and serve as a critical template for 4R tauopathy propagation. These results hint at opportunities for tau isoform-specific therapeutic interventions.
Asunto(s)
Priones , Tauopatías , Humanos , Proteínas tau/metabolismo , Tauopatías/metabolismo , Isoformas de Proteínas/metabolismo , Priones/metabolismo , Péptidos , AminoácidosRESUMEN
The use of variable domain of the heavy-chain of the heavy-chain-only antibodies (VHHs) as disease-modifying biomolecules in neurodegenerative disorders holds promises, including targeting of aggregation-sensitive proteins. Exploitation of their clinical values depends however on the capacity to deliver VHHs with optimal physico-chemical properties for their specific context of use. We described previously a VHH with high therapeutic potential in a family of neurodegenerative diseases called tauopathies. The activity of this promising parent VHH named Z70 relies on its binding within the central region of the tau protein. Accordingly, we carried out random mutagenesis followed by yeast two-hybrid screening to obtain optimized variants. The VHHs selected from this initial screen targeted the same epitope as VHH Z70 as shown using NMR spectroscopy and had indeed improved binding affinities according to dissociation constant values obtained by surface plasmon resonance spectroscopy. The improved affinities can be partially rationalized based on three-dimensional structures and NMR data of three complexes consisting of an optimized VHH and a peptide containing the tau epitope. Interestingly, the ability of the VHH variants to inhibit tau aggregation and seeding could not be predicted from their affinity alone. We indeed showed that the in vitro and in cellulo VHH stabilities are other limiting key factors to their efficacy. Our results demonstrate that only a complete pipeline of experiments, here described, permits a rational selection of optimized VHH variants, resulting in the selection of VHH variants with higher affinities and/or acting against tau seeding in cell models.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Anticuerpos de Dominio Único , Proteínas tau , Humanos , Epítopos/química , Epítopos/inmunología , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/inmunología , Péptidos/química , Péptidos/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunología , Proteínas tau/química , Proteínas tau/inmunologíaRESUMEN
Glycogen synthase kinase 3 (GSK3) plays a pivotal role in signaling pathways involved in insulin metabolism and the pathogenesis of neurodegenerative disorders. In particular, the GSK3ß isoform is implicated in Alzheimer's disease (AD) as one of the key kinases involved in the hyperphosphorylation of tau protein, one of the neuropathological hallmarks of AD. As a constitutively active serine/threonine kinase, GSK3 is inactivated by Akt/PKB-mediated phosphorylation of Ser9 in the N-terminal disordered domain, and for most of its substrates, requires priming (prephosphorylation) by another kinase that targets the substrate to a phosphate-specific pocket near the active site. GSK3 has also been shown to be post-translationally modified by O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation), with still unknown functions. Here, we have found that binding of Akt inhibits GSK3ß kinase activity on both primed and unprimed tau substrates. Akt-mediated Ser9 phosphorylation restores the GSK3ß kinase activity only on primed tau, thereby selectively inactivating GSK3ß toward unprimed tau protein. Additionally, we have shown that GSK3ß is highly O-GlcNAcylated at multiple sites within the kinase domain and the disordered N- and C-terminal domains, including Ser9. In contrast to Akt-mediated regulation, neither the O-GlcNAc transferase nor O-GlcNAcylation significantly alters GSK3ß kinase activity, but high O-GlcNAc levels reduce Ser9 phosphorylation by Akt. Reciprocally, Akt phosphorylation downregulates the overall O-GlcNAcylation of GSK3ß, indicating a crosstalk between both post-translational modifications. Our results indicate that specific O-GlcNAc profiles may be involved in the phosphorylation-dependent Akt-mediated regulation of GSK3ß kinase activity.
Asunto(s)
Glucógeno Sintasa Quinasa 3 beta , Proteínas Proto-Oncogénicas c-akt , Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/química , Fosforilación , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Procesamiento Proteico-Postraduccional , Glucógeno Sintasa Quinasa 3/metabolismo , Acetilglucosamina/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , N-Acetilglucosaminiltransferasas/química , Glicosilación , AnimalesRESUMEN
The identification of chemical starting points for the development of molecular glues is challenging. Here, we employed fragment screening and identified an allosteric stabilizer of the complex between 14-3-3 and a TAZ-derived peptide. The fragment binds preferentially to the 14-3-3/TAZ peptide complex and shows moderate stabilization in differential scanning fluorimetry and microscale thermophoresis. The binding site of the fragment was predicted by molecular dynamics calculations to be distant from the 14-3-3/TAZ peptide interface, located between helices 8 and 9 of the 14-3-3 protein. This site was confirmed by nuclear magnetic resonance and X-ray protein crystallography, revealing the first example of an allosteric stabilizer for 14-3-3 protein-protein interactions.
RESUMEN
RAGE is a transmembrane receptor of immunoglobulin family that can bind various endogenous and exogenous ligands, initiating the inflammatory downstream signaling pathways, including inflammaging. Therefore, RAGE represents an attractive drug target for age-related diseases. For the development of small-molecule RAGE antagonists, we employed protein-templated dynamic combinatorial chemistry (ptDCC) using RAGE's VC1 domain as a template, the first application of this approach in the context of RAGE. The affinities of DCC hits were validated using microscale thermophoresis. Subsequent screening against AGE2 (glyceraldehyde-modified AGE)-sRAGE (solubleRAGE) (AGE2-BSA/sRAGE) interaction using ELISA tests led to the identification of antagonists with micromolar potency. Our findings not only demonstrate the successful application of ptDCC on RAGE but also highlight its potential to address the pressing need for alternative strategies for the development of small-molecule RAGE antagonists, an area of research that has experienced a slowdown in recent years.
Asunto(s)
Transducción de Señal , Receptor para Productos Finales de Glicación Avanzada/química , Receptor para Productos Finales de Glicación Avanzada/metabolismoRESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer , Proteínas tau , Humanos , Proteínas tau/metabolismo , Fosforilación , Dominios Homologos src , Unión Proteica , Enfermedad de Alzheimer/metabolismo , Péptidos/química , Sitios de Unión , Prolina/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
Tau proteins aggregate into filaments in brain cells in Alzheimer's disease and related disorders referred to as tauopathies. Here, we used fragments of camelid heavy-chain-only antibodies (VHHs or single domain antibody fragments) targeting Tau as immuno-modulators of its pathologic seeding. A VHH issued from the screen against Tau of a synthetic phage-display library of humanized VHHs was selected for its capacity to bind Tau microtubule-binding domain, composing the core of Tau fibrils. This parent VHH was optimized to improve its biochemical properties and to act in the intra-cellular compartment, resulting in VHH Z70. VHH Z70 precisely binds the PHF6 sequence, known for its nucleation capacity, as shown by the crystal structure of the complex. VHH Z70 was more efficient than the parent VHH to inhibit in vitro Tau aggregation in heparin-induced assays. Expression of VHH Z70 in a cellular model of Tau seeding also decreased the aggregation-reporting fluorescence signal. Finally, intra-cellular expression of VHH Z70 in the brain of an established tauopathy mouse seeding model demonstrated its capacity to mitigate accumulation of pathological Tau. VHH Z70, by targeting Tau inside brain neurons, where most of the pathological Tau resides, provides an immunological tool to target the intra-cellular compartment in tauopathies.
Asunto(s)
Enfermedad de Alzheimer , Anticuerpos de Dominio Único , Tauopatías , Enfermedad de Alzheimer/metabolismo , Animales , Modelos Animales de Enfermedad , Ratones , Neuronas/metabolismo , Proteínas Represoras , Tauopatías/metabolismo , Proteínas tau/genéticaRESUMEN
The main protease (3CLp) of the SARS-CoV-2, the causative agent for the COVID-19 pandemic, is one of the main targets for drug development. To be active, 3CLp relies on a complex interplay between dimerization, active site flexibility, and allosteric regulation. The deciphering of these mechanisms is a crucial step to enable the search for inhibitors. In this context, using NMR spectroscopy, we studied the conformation of dimeric 3CLp from the SARS-CoV-2 and monitored ligand binding, based on NMR signal assignments. We performed a fragment-based screening that led to the identification of 38 fragment hits. Their binding sites showed three hotspots on 3CLp, two in the substrate binding pocket and one at the dimer interface. F01 is a non-covalent inhibitor of the 3CLp and has antiviral activity in SARS-CoV-2 infected cells. This study sheds light on the complex structure-function relationships of 3CLp and constitutes a strong basis to assist in developing potent 3CLp inhibitors.
Asunto(s)
Antivirales/farmacología , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/farmacología , SARS-CoV-2/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Antivirales/química , Sitios de Unión , Chlorocebus aethiops , Proteasas 3C de Coronavirus/química , Inhibidores de Cisteína Proteinasa/química , Evaluación Preclínica de Medicamentos , Pruebas de Sensibilidad Microbiana , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Multimerización de Proteína , SARS-CoV-2/química , Bibliotecas de Moléculas Pequeñas/química , Células VeroRESUMEN
Natural compounds are an important class of potent drug molecules including some retrospectively found to act as stabilizers of protein-protein interactions (PPIs). However, the design of synthetic PPI stabilizers remains an understudied approach. To date, there are limited examples where cooperativity has been utilized to guide the optimization of a PPI stabilizer. The 14-3-3 scaffold proteins provide an excellent platform to explore PPI stabilization because these proteins mediate several hundred PPIs, and a class of natural compounds, the fusicoccanes, are known to stabilize a subset of 14-3-3 protein interactions. 14-3-3 has been reported to negatively regulate the p65 subunit of the NF-κB transcription factor, which qualifies this protein complex as a potential target for drug discovery to control cell proliferation. Here, we report the high-resolution crystal structures of two 14-3-3 binding motifs of p65 in complex with 14-3-3. A semisynthetic natural product derivative, DP-005, binds to an interface pocket of the p65/14-3-3 complex and concomitantly stabilizes it. Cooperativity analyses of this interaction, and other disease relevant 14-3-3-PPIs, demonstrated selectivity of DP-005 for the p65/14-3-3 complex. The adaptation of a cooperative binding model provided a general approach to characterize stabilization and to assay for selectivity of PPI stabilizers.
Asunto(s)
Proteínas 14-3-3/química , Productos Biológicos/química , FN-kappa B/química , Productos Biológicos/síntesis química , Humanos , Modelos Moleculares , Unión ProteicaRESUMEN
Protein-protein interactions (PPIs) of 14-3-3 proteins are a model system for studying PPI stabilization. The complex natural product Fusicoccinâ A stabilizes many 14-3-3 PPIs but is not amenable for use in SAR studies, motivating the search for more drug-like chemical matter. However, drug-like 14-3-3 PPI stabilizers enabling such studies have remained elusive. An X-ray crystal structure of a PPI in complex with an extremely low potency stabilizer uncovered an unexpected non-protein interacting, ligand-chelated Mg2+ leading to the discovery of metal-ion-dependent 14-3-3 PPI stabilization potency. This originates from a novel chelation-controlled bioactive conformation stabilization effect. Metal chelation has been associated with pan-assay interference compounds (PAINS) and frequent hitter behavior, but chelation can evidently also lead to true potency gains and find use as a medicinal chemistry strategy to guide compound optimization. To demonstrate this, we exploited the effect to design the first potent, selective, and drug-like 14-3-3 PPI stabilizers.
Asunto(s)
Proteínas 14-3-3/química , Productos Biológicos/química , Quelantes/química , Metales/química , Descubrimiento de Drogas , Glicósidos , Humanos , Conformación Molecular , Unión ProteicaRESUMEN
Tauopathies are neurodegenerative diseases characterized by the intraneuronal accumulation of aggregated tau. The staging of this neurodegenerative process is well established for Alzheimer's disease as well as for other tauopathies. The stereotypical pattern of tau pathology in these diseases is consistent with the hypothesis that the tau protein can spread in a 'prion-like' manner. It proposes that extracellular pathological tau species can transmit pathology from cell to cell. Accordingly, by targeting these spreading species with therapeutic antibodies one should be able to slow or halt the progression of tau pathology. To be effective, antibodies should neutralize the pathological species present in Alzheimer's disease brains and block their cell-to-cell spread. To evaluate both aspects, tau antibody D, which recognizes an epitope in the central region of tau, and was selected for its outstanding ability to block tau seeding in cell based assays, was used in this study. Here, we addressed two fundamental questions: (i) can this anti-tau antibody neutralize the pathological species present in Alzheimer's disease brains; and (ii) can it block the cell-to-cell spread of tau seeds in vivo? First, antibody D effectively prevented the induction of tau pathology in the brains of transgenic mice that had been injected with human Alzheimer's disease brain extracts, showing that it could effectively neutralize the pathological species present in these extracts. Second, by using K18 P301L tau fibrils to induce pathology, we further demonstrated that antibody D was also capable of blocking the progression of tau pathology to distal brain regions. In contrast, an amino-terminal tau antibody, which was less effective at blocking tau seeding in vitro showed less efficacy in reducing Alzheimer's disease patient tau driven pathology in the transgenic mouse model. We did not address whether the same is true for a spectrum of other amino-terminal antibodies that were tested in vitro. These data highlight important differences between tau antibodies and, when taken together with other recently published data, suggest that epitope may be important for function.
Asunto(s)
Enfermedad de Alzheimer/patología , Ovillos Neurofibrilares/patología , Tauopatías/metabolismo , Proteínas tau/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/terapia , Animales , Anticuerpos/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Epítopos , Femenino , Factores Inmunológicos/metabolismo , Inmunoterapia , Masculino , Ratones Transgénicos , Proteínas tau/metabolismoRESUMEN
Accumulating data support the role of tau pathology in cognitive decline in ageing and Alzheimer's disease, but underlying mechanisms remain ill-defined. Interestingly, ageing and Alzheimer's disease have been associated with an abnormal upregulation of adenosine A2A receptor (A2AR), a fine tuner of synaptic plasticity. However, the link between A2AR signalling and tau pathology has remained largely unexplored. In the present study, we report for the first time a significant upregulation of A2AR in patients suffering from frontotemporal lobar degeneration with the MAPT P301L mutation. To model these alterations, we induced neuronal A2AR upregulation in a tauopathy mouse model (THY-Tau22) using a new conditional strain allowing forebrain overexpression of the receptor. We found that neuronal A2AR upregulation increases tau hyperphosphorylation, potentiating the onset of tau-induced memory deficits. This detrimental effect was linked to a singular microglial signature as revealed by RNA sequencing analysis. In particular, we found that A2AR overexpression in THY-Tau22 mice led to the hippocampal upregulation of C1q complement protein-also observed in patients with frontotemporal lobar degeneration-and correlated with the loss of glutamatergic synapses, likely underlying the observed memory deficits. These data reveal a key impact of overactive neuronal A2AR in the onset of synaptic loss in tauopathies, paving the way for new therapeutic approaches.
Asunto(s)
Complemento C1q/metabolismo , Neuronas/metabolismo , Receptor de Adenosina A2A/genética , Receptor de Adenosina A2A/metabolismo , Sinapsis/patología , Tauopatías/genética , Tauopatías/patología , Animales , Autopsia , Degeneración Lobar Frontotemporal/genética , Degeneración Lobar Frontotemporal/metabolismo , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Trastornos de la Memoria/etiología , Trastornos de la Memoria/psicología , Ratones , Ratones Transgénicos , Mutación , Aprendizaje Espacial , Tauopatías/psicología , Proteínas tau/genéticaRESUMEN
Determining the functional relationship between Tau phosphorylation and aggregation has proven a challenge owing to the multiple potential phosphorylation sites and their clustering in the Tau sequence. We use here in vitro kinase assays combined with NMR spectroscopy as an analytical tool to generate well-characterized phosphorylated Tau samples and show that the combined phosphorylation at the Ser202/Thr205/Ser208 sites, together with absence of phosphorylation at the Ser262 site, yields a Tau sample that readily forms fibers, as observed by thioflavin T fluorescence and electron microscopy. On the basis of conformational analysis of synthetic phosphorylated peptides, we show that aggregation of the samples correlates with destabilization of the turn-like structure defined by phosphorylation of Ser202/Thr205.
Asunto(s)
Agregación Patológica de Proteínas , Serina/metabolismo , Treonina/metabolismo , Proteínas tau/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Humanos , Espectroscopía de Resonancia Magnética , Microscopía Electrónica de Transmisión , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/ultraestructura , Fosforilación , Dominios Proteicos , Ratas Sprague-Dawley , Serina/química , Serina/genética , Treonina/química , Treonina/genética , Proteínas tau/química , Proteínas tau/genéticaRESUMEN
The bridging integrator 1 gene (BIN1) is a major genetic risk factor for Alzheimer's disease (AD). In this report, we investigated how BIN1-dependent pathophysiological processes might be associated with Tau. We first generated a cohort of control and transgenic mice either overexpressing human MAPT (TgMAPT) or both human MAPT and BIN1 (TgMAPT;TgBIN1), which we followed-up from 3 to 15 months. In TgMAPT;TgBIN1 mice short-term memory deficits appeared earlier than in TgMAPT mice; however-unlike TgMAPT mice-TgMAPT;TgBIN1 mice did not exhibit any long-term or spatial memory deficits for at least 15 months. After killing the cohort at 18 months, immunohistochemistry revealed that BIN1 overexpression prevents both Tau mislocalization and somatic inclusion in the hippocampus, where an increase in BIN1-Tau interaction was also observed. We then sought mechanisms controlling the BIN1-Tau interaction. We developed a high-content screening approach to characterize modulators of the BIN1-Tau interaction in an agnostic way (1,126 compounds targeting multiple pathways), and we identified-among others-an inhibitor of calcineurin, a Ser/Thr phosphatase. We determined that calcineurin dephosphorylates BIN1 on a cyclin-dependent kinase phosphorylation site at T348, promoting the open conformation of the neuronal BIN1 isoform. Phosphorylation of this site increases the availability of the BIN1 SH3 domain for Tau interaction, as demonstrated by nuclear magnetic resonance experiments and in primary neurons. Finally, we observed that although the levels of the neuronal BIN1 isoform were unchanged in AD brains, phospho-BIN1(T348):BIN1 ratio was increased, suggesting a compensatory mechanism. In conclusion, our data support the idea that BIN1 modulates the AD risk through an intricate regulation of its interaction with Tau. Alteration in BIN1 expression or activity may disrupt this regulatory balance with Tau and have direct effects on learning and memory.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Trastornos de la Memoria/metabolismo , Memoria a Largo Plazo/fisiología , Proteínas del Tejido Nervioso/metabolismo , Tauopatías/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas tau/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Trastornos de la Memoria/genética , Trastornos de la Memoria/patología , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Neuronas/patología , Fosforilación , Memoria Espacial/fisiología , Tauopatías/genética , Tauopatías/patología , Proteínas Supresoras de Tumor/genéticaRESUMEN
Although Tau is an intrinsically disordered protein, some level of structure can still be defined, corresponding to short stretches of dynamic secondary structures and a preferential global fold described as an ensemble of conformations. These structures can be modified by Tau phosphorylation, and potentially other post-translational modifications. The analytical capacity of Nuclear Magnetic Resonance (NMR) spectroscopy provides the advantage of offering a residue-specific view of these modifications, allowing to link specific sites to a particular structure. The cis or trans conformation of X-Proline peptide bonds is an additional characteristic parameter of Tau structure that is targeted and modified by prolyl cis/trans isomerases. The challenge in molecular characterization of Tau lies in being able to link structural parameters to functional consequences in normal functions and dysfunctions of Tau, including potential misfolding on the path to aggregation and/or perturbation of the interactions of Tau with its many molecular partners. Phosphorylation of Ser and Thr residues has the potential to impact the local and global structure of Tau.
Asunto(s)
Procesamiento Proteico-Postraduccional , Proteínas tau/química , Proteínas tau/metabolismo , Humanos , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Estructura Secundaria de ProteínaRESUMEN
Metal ions are well known modulators of protein aggregation and are key players in Alzheimer's Disease, being found to be associated to pathologic protein deposits in diseased brains. Therefore, understanding how metals influence amyloid aggregation is critical in establishing molecular mechanisms that underlie disease onset and progression. Here, we report data on the interaction of full-length human Tau protein with calcium and zinc ions, evidencing that Tau self-assembly is differently regulated, depending on the type of bound metal ion. We established that Tau binds 4 Zn2+ and 1 Ca2+ per monomer while using native mass spectrometry analysis, without inducing order or substantial conformational changes in the intrinsically disordered Tau, as determined by structural analysis using circular dichroism and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopies. However, Tau aggregation is found to proceed differently in the calcium- and -zinc bound forms. While the rate of aggregation, as determined from thioflavin-T (ThT) fluorescence kinetics, is highly increased in both cases, the reaction proceeds via different mechanisms, as evidenced by the absence of the lag phase in the reaction of zinc-bound Tau. Monitoring Tau aggregation using native mass spectrometry indeed evidenced a distinct distribution of Tau conformers along the reaction, as confirmed by dynamic light scattering analysis. We propose that such differences arise from zinc binding at distinct locations within the Tau sequence that prompt both the rapid formation of seeding oligomers through interactions at high affinity sites within the repeat domains, as well as amorphous aggregation, through low affinity interactions with residues elsewhere in the sequence, including at the fuzzy coat domain.
Asunto(s)
Agregado de Proteínas/fisiología , Zinc/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/metabolismo , Amiloide/metabolismo , Benzotiazoles/metabolismo , Calcio/metabolismo , Dicroismo Circular , Humanos , Cinética , Conformación Proteica , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
Non-structural protein 5B (NS5B) is the RNA-dependent RNA polymerase that catalyzes replication of the hepatitis C virus (HCV) RNA genome and therefore is central for its life cycle. NS5B interacts with the intrinsically disordered domain 2 of NS5A (NS5A-D2), another essential multifunctional HCV protein that is required for RNA replication. As a result, these two proteins represent important targets for anti-HCV chemotherapies. Despite this importance and the existence of NS5B crystal structures, our understanding of the conformational and dynamic behavior of NS5B in solution and its relationship with NS5A-D2 remains incomplete. To address these points, we report the first detailed NMR spectroscopic study of HCV NS5B lacking its membrane anchor (NS5BΔ21). Analysis of constructs with selective isotope labeling of the δ1 methyl groups of isoleucine side chains demonstrates that, in solution, NS5BΔ21 is highly dynamic but predominantly adopts a closed conformation. The addition of NS5A-D2 leads to spectral changes indicative of binding to both allosteric thumb sites I and II of NS5BΔ21 and induces long-range perturbations that affect the RNA-binding properties of the polymerase. We compared these modifications with the short- and long-range effects triggered in NS5BΔ21 upon binding of filibuvir, an allosteric inhibitor. We demonstrate that filibuvir-bound NS5BΔ21 is strongly impaired in the binding of both NS5A-D2 and RNA. NS5A-D2 induces conformational and functional perturbations in NS5B similar to those triggered by filibuvir. Thus, our work highlights NS5A-D2 as an allosteric regulator of the HCV polymerase and provides new insight into the dynamics of NS5B in solution.
Asunto(s)
Hepacivirus/enzimología , Modelos Moleculares , Oligorribonucleótidos/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas no Estructurales Virales/metabolismo , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico/efectos de los fármacos , Antivirales/química , Antivirales/metabolismo , Antivirales/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Eliminación de Gen , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Isoleucina/química , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Oligorribonucleótidos/química , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Mutación Puntual , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Replegamiento Proteico/efectos de los fármacos , Pironas/química , Pironas/metabolismo , Pironas/farmacología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidad , Triazoles/química , Triazoles/metabolismo , Triazoles/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genéticaRESUMEN
Lamin B Receptor (LBR) is an integral protein of the interphase inner nuclear membrane that is implicated in chromatin anchorage to the nuclear envelope. Phosphorylation of a stretch of arginine-serine (RS) dipeptides in the amino-terminal nucleoplasmic domain of LBR regulates the interactions of the receptor with other nuclear proteins, DNA and RNA and thus modulates tethering of heterochromatin to the nuclear envelope. While phosphorylation has been extensively studied, very little is known about other post-translational modifications of the protein. There is only one report on the O-ß-linked N-acetyl-glucosaminylation (O-GlcNAcylation) of a serine residue downstream of the RS domain of rat LBR. In the present study we identify additional O-GlcNAcylation sites by using as substrates of O-ß-N-acetylglucosaminyltransferase (OGT) a set of peptides containing the entire LBR RS domain or parts of it as well as flanking sequences. The in vitro activity of OGT was assessed by tandem mass spectrometry and NMR spectroscopy. Furthermore, we provide evidence that O-GlcNAcylation hampers DNA binding while it marginally affects RS domain phosphorylation mediated by SRPK1, Akt2 and cdk1 kinases. GENERAL SIGNIFICANCE: Our methodology providing a quantitative description of O-GlcNAc patterns based on a combination of mass spectrometry and high resolution NMR spectroscopy on short peptide substrates allows subsequent functional analyses. Hence, our approach is of general interest to a wide audience of biologists aiming at deciphering the functional role of O-GlcNAc glycosylation and its crosstalk with phosphorylation.
Asunto(s)
Acetilglucosamina/metabolismo , ADN/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , ADN/genética , Glicosilación , Humanos , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Péptidos/genética , Péptidos/metabolismo , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Homología de Secuencia de Aminoácido , Pavos , Receptor de Lamina BRESUMEN
Tau neuronal protein has a central role in neurodegeneration and is implicated in Alzheimer disease development. Abnormal phosphorylation of Tau impairs its interaction with other proteins and is associated with its dysregulation in pathological conditions. Molecular mechanisms leading to hyperphosphorylation of Tau in pathological conditions are unknown. Here, we characterize phosphorylation of Tau by extracellular-regulated kinase (ERK2), a mitogen-activated kinase (MAPK) that responds to extracellular signals. Analysis ofin vitrophosphorylated Tau by activated recombinant ERK2 with nuclear magnetic resonance spectroscopy (NMR) reveals phosphorylation of 15 Ser/Thr sites.In vitrophosphorylation of Tau using rat brain extract and subsequent NMR analysis identifies the same sites. Phosphorylation with rat brain extract is known to transform Tau into an Alzheimer disease-like state. Our results indicate that phosphorylation of Tau by ERK2 alone is sufficient to produce the same characteristics. We further investigate the mechanism of ERK2 phosphorylation of Tau. Kinases are known to recognize their protein substrates not only by their specificity for a targeted Ser or Thr phosphorylation site but also by binding to linear-peptide motifs called docking sites. We identify two main ERK2 docking sites in Tau sequence using NMR. Our results suggest that ERK2 dysregulation in Alzheimer disease could lead to abnormal phosphorylation of Tau resulting in the pathology of the disease.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/química , Proteínas tau/química , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Secuencias de Aminoácidos , Animales , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Estructura Terciaria de Proteína , Ratas , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
The N-terminal acidic transactivation domain (TAD) of ERM/ETV5 (ERM38-68), a PEA3 group member of Ets-related transcription factors, directly interacts with the ACID/PTOV domain of the Mediator complex subunit MED25. Molecular details of this interaction were investigated using nuclear magnetic resonance (NMR) spectroscopy. The TAD is disordered in solution but has a propensity to adopt local transient secondary structure. We show that it folds upon binding to MED25 and that the resulting ERM-MED25 complex displays characteristics of a fuzzy complex. Mutational analysis further reveals that two aromatic residues in the ERM TAD (F47 and W57) are involved in the binding to MED25 and participate in the ability of ERM TAD to activate transcription. Mutation of a key residue Q451 in the VP16 H1 binding pocket of MED25 affects the binding of ERM. Furthermore, competition experiments show that ERM and VP16 H1 share a common binding interface on MED25. NMR data confirms the occupancy of this binding pocket by ERM TAD. Based on these experimental data, a structural model of a functional interaction is proposed. This study provides mechanistic insights into the Mediator-transactivator interactions.