RESUMO
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.
Assuntos
Ubiquitina , Ubiquitinação , Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/química , Humanos , Ubiquitina/metabolismo , Ubiquitina/química , Estrutura MolecularRESUMO
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
BACKGROUND: Biosurfactants are surface-active compounds with environmental and industrial applications. These molecules show higher biocompatibility, stability and efficiency compared to synthetic surfactants. On the other hand, biosurfactants are not cost-competitive to their chemical counterparts. Cost effective technology such as the use of low-cost substrates is a promising approach aimed at reducing the production cost. This study aimed to evaluate the biosurfactant production and activity by the novel strain Rhodococcus sp. SP1d by using different growth substrates. Therefore, to exploit the biosurfactant synthesized by SP1d for environmental applications, the effect of this compound on the bacteria biofilm formation was evaluated. Eventually, for a possible bioremediation application, the biosurfactant properties and its chemical characteristics were investigated using diesel as source of carbon. RESULTS: Rhodococcus sp. SP1d evidence the highest similarity to Rhodococcus globerulus DSM 43954T and the ability to biosynthesize surfactants using a wide range of substrates such as exhausted vegetable oil, mineral oil, butter, n-hexadecane, and diesel. The maximum production of crude biosurfactant after 10 days of incubation was reached on n-hexadecane and diesel with a final yield of 2.38 ± 0.51 and 1.86 ± 0.31 g L- 1 respectively. Biosurfactants produced by SP1d enhanced the biofilm production of P. protegens MP12. Moreover, the results showed the ability of SP1d to produce biosurfactants on diesel even when grown at 10 and 18 °C. The biosurfactant activity was maintained over a wide range of NaCl concentration, pH, and temperature. A concentration of 1000 mg L- 1 of the crude biosurfactant showed an emulsification activity of 55% towards both xylene and olive oil and a reduction of 25.0 mN m- 1 of surface tension of water. Eventually, nuclear magnetic resonance spectroscopy indicated that the biosurfactant is formed by trehalolipids. CONCLUSIONS: The use of low-cost substrates such as exhausted oils and waste butter reduce both the costs of biosurfactant synthesis and the environmental pollution due to the inappropriate disposal of these residues. High production yields, stability and emulsification properties using diesel and n-hexadecane as substrates, make the biosurfactant produced by SP1d a sustainable biocompound for bioremediation purpose. Eventually, the purified biosurfactant improved the biofilm formation of the fungal antagonistic strain P. protegens MP12, and thus seem to be exploitable to increase the adherence and colonization of plant surfaces by this antagonistic strain and possibly enhance antifungal activity.
Assuntos
Alcanos , Rhodococcus , Tensoativos/química , Tensão Superficial , Biodegradação AmbientalRESUMO
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
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
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
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.
Assuntos
Doença de Alzheimer , Tauopatias , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Proteínas tau/metabolismo , UbiquitinaçãoRESUMO
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
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.
Assuntos
Ubiquitina-Proteína Ligases , Proteínas tau , Chaperonas Moleculares/metabolismo , Ubiquitina/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas tau/metabolismoRESUMO
The extraordinary flexibility and structural heterogeneity of intrinsically disordered proteins (IDP) make them functionally versatile molecules. We have now begun to better understand their fundamental role in biology, however many aspects of their behaviour remain difficult to grasp experimentally. This is especially true for the intermolecular interactions which lead to the formation of transient or highly dynamic supramolecular self-assemblies, such as oligomers, aggregation intermediates and biomolecular condensates. Both the emerging functions and pathogenicity of these structures have stimulated great efforts to develop methodologies capable of providing useful insights. Significant progress in solution NMR spectroscopy has made this technique one of the most powerful to describe structural and dynamic features of IDPs within such assemblies at atomic resolution. Here, we review the most recent works that have illuminated key aspects of IDP assemblies and contributed significant advancements towards our understanding of the complex conformational landscape of prototypical disease-associated proteins. We also include a primer on some of the fundamental and innovative NMR methods being used in the discussed studies.
Assuntos
Proteínas Amiloidogênicas/química , Proteínas Intrinsicamente Desordenadas/química , Espectroscopia de Ressonância Magnética , Adsorção , Humanos , Proteína Huntingtina/química , Cinética , Substâncias Macromoleculares , Peptídeos/química , Ligação Proteica , Conformação Proteica , Proteínas tau/químicaRESUMO
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
Alpha-synuclein (αS) is an extensively studied protein due to its involvement in a group of neurodegenerative disorders, including Parkinson's disease, and its documented ability to undergo aberrant self-aggregation resulting in the formation of amyloid-like fibrils. In dilute solution, the protein is intrinsically disordered but can adopt multiple alternative conformations under given conditions, such as upon adsorption to nanoscale surfaces. The study of αS-nanoparticle interactions allows us to better understand the behavior of the protein and provides the basis for developing systems capable of mitigating the formation of toxic aggregates as well as for designing hybrid nanomaterials with novel functionalities for applications in various research areas. In this review, we summarize current progress on αS-nanoparticle interactions with an emphasis on the conformational plasticity of the biomolecule.
Assuntos
Nanopartículas/química , Nanopartículas/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Adsorção , Amiloide , Humanos , Conformação Molecular , Nanoconjugados/química , Agregados ProteicosRESUMO
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
In the brain of individuals with Alzheimer's disease, the regulatory protein ubiquitin is found conjugated to different lysine residues of tau protein assembled into pathological paired helical filaments. To shed light on the hitherto unexplored ubiquitination-linked conformational transitions of tau, the availability of in vitro ubiquitin conjugation methods is of primary importance. In our work, we focused on the four-repeat domain of tau and assembled an enzymatic machinery formed by UBE1, Ubc13, and CHIP enzymes. The enzymatic reaction resulted in monoubiquitination at multiple sites, reminiscent of the ubiquitination pattern observed in vivo. We further exploited chemoselective disulfide coupling reactions to construct three tau regioisomers with site-specific monoubiquitination. Protein aggregation experiments revealed that the multiple enzyme-derived products were unable to convert into amyloid fibrils, while the semisynthetic conjugates exhibited diverse capability to form filaments. This study contributes novel insight into the effects of a key post-translational modification on aberrant protein self-assembly.
Assuntos
Peptídeos/metabolismo , Agregados Proteicos , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas tau/química , Sequência de Aminoácidos , Amiloide/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Peptídeos/química , Estereoisomerismo , Ubiquitinação , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Calmodulin-like (CML) proteins are major EF-hand-containing, calcium (Ca2+)-binding proteins with crucial roles in plant development and in coordinating plant stress tolerance. Given their abundance in plants, the properties of Ca2+ sensors and identification of novel target proteins of CMLs deserve special attention. To this end, we recombinantly produced and biochemically characterized CML36 from Arabidopsis thaliana We analyzed Ca2+ and Mg2+ binding to the individual EF-hands, observed metal-induced conformational changes, and identified a physiologically relevant target. CML36 possesses two high-affinity Ca2+/Mg2+ mixed binding sites and two low-affinity Ca2+-specific sites. Binding of Ca2+ induced an increase in the α-helical content and a conformational change that lead to the exposure of hydrophobic regions responsible for target protein recognition. Cation binding, either Ca2+ or Mg2+, stabilized the secondary and tertiary structures of CML36, guiding a large structural transition from a molten globule apo-state to a compact holoconformation. Importantly, through in vitro binding and activity assays, we showed that CML36 interacts directly with the regulative N terminus of the Arabidopsis plasma membrane Ca2+-ATPase isoform 8 (ACA8) and that this interaction stimulates ACA8 activity. Gene expression analysis revealed that CML36 and ACA8 are co-expressed mainly in inflorescences. Collectively, our results support a role for CML36 as a Ca2+ sensor that binds to and modulates ACA8, uncovering a possible involvement of the CML protein family in the modulation of plant-autoinhibited Ca2+ pumps.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/química , ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Membrana Celular/enzimologia , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Calmodulina/genética , Ativação EnzimáticaRESUMO
In biological systems, nanoparticles (NPs) elicit bioactivity upon interaction with proteins. As a result of post-translational modification, proteins occur in a variety of alternative covalent forms, including structural isomers, which present unique molecular surfaces. We aimed at a detailed description of the recognition of protein isomeric species by NP surfaces. The transient adsorption of isomeric ubiquitin (Ub) dimers by NPs was investigated by solution NMR spectroscopy. Lys63- and Lys48-linked Ub2 were adsorbed by large anionic NPs with different affinities, whereas the binding strength was similar in the cases of smaller particles. After the incorporation of paramagnetic tags into NPs, the observed site-resolved paramagnetic footprints provided a high-resolution map of the different protein surfaces binding to NPs. The approach described could be extended to further protein isoforms and more specialized NP systems to allow better control of the interactions between NPs and protein targets.
Assuntos
Nanopartículas/química , Proteínas/química , Ubiquitina/química , Adsorção , Isomerismo , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
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.
Assuntos
Proteínas de Ligação a Ácido Graxo/química , Ácido Glicocólico/metabolismo , Ácido Oleico/metabolismo , Polimorfismo de Nucleotídeo Único , Regulação Alostérica , Substituição de Aminoácidos , Sítios de Ligação , Proteínas de Ligação a Ácido Graxo/genética , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/metabolismoRESUMO
Ferritin is a ubiquitous nanocage protein, which can accommodate up to thousands of iron atoms inside its cavity. Aside from its iron storage function, a new role as a fatty acid binder has been proposed for this protein. The interaction of apo horse spleen ferritin (HoSF) with a variety of lipids has been here investigated through NMR spectroscopic ligand-based experiments, to provide new insights into the mechanism of ferritin-lipid interactions, and the link with iron mineralization. 1D 1 H,â diffusion (DOSY) and saturation-transfer difference (STD)â NMR experiments provided evidence for a stronger interaction of ferritin with unsaturated fatty acids compared to saturated fatty acids, detergents, and bile acids. Mineralization assays showed that oleate c aused the most efficient increase in the initial rate of iron oxidation, and the highest formation of ferric species in HoSF. The comprehension of the factors inducing a faster biomineralization is an issue of the utmost importance, given the association of ferritin levels with metabolic syndromes, such as insulin resistance and diabetes, characterized by fatty acid concentration dysregulation. The human ferritin H-chain homopolymer (HuHF), featuring ferroxidase activity, was also tested for its fatty acid binding capabilities. Assays show that oleate can bind with high affinity to HuHF, without altering the reaction rates at the ferroxidase site.
Assuntos
Ácidos Graxos Insaturados/química , Ferritinas/química , Ferro/metabolismo , Animais , Apoproteínas/química , Apoproteínas/metabolismo , Ceruloplasmina/química , Ceruloplasmina/metabolismo , Cromatografia em Gel , Dicroísmo Circular , Difusão Dinâmica da Luz , Ferritinas/metabolismo , Cavalos , Humanos , Ferro/química , Ligantes , Espectroscopia de Ressonância Magnética , Concentração Osmolar , Ligação ProteicaRESUMO
BACKGROUND: Ileal bile acid-binding protein, IBABP, participates in the intracellular trafficking of bile salts and influences their signaling activities. The recently discovered variant, IBABP-L, bearing an N-terminal 49-amino acid extension, was found to be associated with colorectal cancer and to protect cancer cells from the cytotoxic effects of deoxycholate. However, the precise function and the molecular properties of this variant are currently unknown. METHODS: Bioinformatics tools and confocal microscopy were used to investigate the sub-cellular localization of IBABP-L; protein dynamics, ligand binding and interaction with membrane models were studied by 2D NMR and fluorescence spectroscopy. RESULTS: Based on sub-cellular localization experiments we conclude that IBABP-L is targeted to the secretory pathway by a 24-residue signal peptide and, upon its cleavage, the mature protein is constitutively released into the extracellular space. Site-resolved NMR experiments indicated the distinct preference of primary and secondary bile salts to form either heterotypic or homotypic complexes with IBABP-L. The presence of the relatively dynamic N-terminal extension, originating only subtle conformational perturbations in the globular domain, was found to influence binding site occupation in IBABP-L as compared to IBABP. Even more pronounced differences were found in the tendency of the two variants to associate with phospholipid bilayers. CONCLUSIONS: IBABP-L exhibits different sub-cellular localization, ligand-binding properties and membrane interaction propensity compared to the canonical short isoform. GENERAL SIGNIFICANCE: Our results constitute an essential first step towards an understanding of the role of IBABP-L in bile salt trafficking and signaling under healthy and pathological conditions.
Assuntos
Proteínas de Transporte/análise , Neoplasias Colorretais/etiologia , Íleo/metabolismo , Bicamadas Lipídicas/metabolismo , Glicoproteínas de Membrana/análise , Ácidos e Sais Biliares/metabolismo , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Células HEK293 , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Isoformas de ProteínasRESUMO
The successful application of nanomaterials in biosciences necessitates an in-depth understanding of how they interface with biomolecules. Transient associations of proteins with nanoparticles (NPs) are accessible by solution NMR spectroscopy, albeit with some limitations. The incorporation of paramagnetic centers into NPs offers new opportunities to explore bio-nano interfaces. We propose NMR paramagnetic relaxation enhancement as a new tool to detect NP-binding surfaces on proteins with increased sensitivity, also extending the applicability of NMR investigations to heterogeneous biomolecular mixtures. The adsorption of ubiquitin on gadolinium-doped fluoride-based NPs produced residue-specific NMR line-broadening effects mapping to a contiguous area on the surface of the protein. Importantly, an identical paramagnetic fingerprint was observed in the presence of a competing protein-protein association equilibrium, exemplifying possible interactions taking place in crowded biological media. The interaction was further characterized using isothermal titration calorimetry and upconversion emission measurements. The data indicate that the used fluoride-based NPs are not biologically inert but rather are capable of biomolecular recognition.