RESUMO
Metal micronutrients are essential for life and exist in a delicate balance to maintain an organism's health. The labile nature of metal-biomolecule interactions clouds the understanding of metal binders and metal-mediated conformational changes that are influential to health and disease. Mass spectrometry (MS)-based methods and technologies have been developed to better understand metal micronutrient dynamics in the intra- and extracellular environment. In this review, we describe the challenges associated with studying labile metals in human biology and highlight MS-based methods for the discovery and study of metal-biomolecule interactions.
Assuntos
Metais , Humanos , Metais/química , Espectrometria de Massas/métodosRESUMO
Transition metal dysregulation is associated with a host of pathologies, many of which are therapeutically targeted using chelators and ionophores. Chelators and ionophores are used as therapeutic metal-binding compounds which impart biological effects by sequestering or trafficking endogenous metal ions in an effort to restore homeostasis. Many current therapies take inspiration or derive directly from small molecules and peptides found in plants. This review focuses on plant-derived small molecule and peptide chelators and ionophores that can affect metabolic disease states. Understanding the coordination chemistry, bioavailability, and bioactivity of such molecules provides the tools to further research applications of plant-based chelators and ionophores.
Assuntos
Quelantes , Elementos de Transição , Ionóforos/farmacologia , Ionóforos/uso terapêutico , Ionóforos/química , Quelantes/farmacologia , Quelantes/uso terapêutico , Quelantes/química , Metais , Plantas/metabolismo , PeptídeosRESUMO
Peptide hormones are essential signaling molecules with therapeutic importance. Identifying regulatory factors that drive their activity gives important insight into their mode of action and clinical development. In this work, we demonstrate the combined impact of Cu(II) and the serum protein albumin on the activity of C-peptide, a 31-mer peptide derived from the same prohormone as insulin. C-peptide exhibits beneficial effects, particularly in diabetic patients, but its clinical use has been hampered by a lack of mechanistic understanding. We show that Cu(II) mediates the formation of ternary complexes between albumin and C-peptide and that the resulting species depend on the order of addition. These ternary complexes notably alter peptide activity, showing differences from the peptide or Cu(II)/peptide complexes alone in redox protection as well as in cellular internalization of the peptide. In standard clinical immunoassays for measuring C-peptide levels, the complexes inflate the quantitation of the peptide, suggesting that such adducts may affect biomarker quantitation. Altogether, our work points to the potential relevance of Cu(II)-linked C-peptide/albumin complexes in the peptide's mechanism of action and application as a biomarker.
Assuntos
Cobre , Albumina Sérica , Humanos , Albumina Sérica/metabolismo , Cobre/química , Peptídeo C , Peptídeos/metabolismo , OxirreduçãoRESUMO
The development of bioluminescence-based tools has seen steady growth in the field of chemical biology over the past few decades ranging in uses from reporter genes to assay development and targeted imaging. More recently, coelenterazine-utilizing luciferases such as Gaussia, Renilla, and the engineered nano-luciferases have been utilized due to their intense luminescence relative to firefly luciferin/luciferase. The emerging importance of these systems warrants investigations into the components that affect their light production. Previous work has reported that one marine luciferase, Gaussia, is potently inhibited by copper salt. The mechanism for inhibition was not elucidated but was hypothesized to occur via binding to the enzyme. In this study, we provide the first report of a group of nonhomologous marine luciferases also exhibiting marked decreases in light emission in the presence of copper (II). We investigate the mechanism of action behind this inhibition and demonstrate that the observed copper inhibition does not stem from a luciferase interaction but rather the chemical oxidation of imidazopyrazinone luciferins generating inert, dehydrated luciferins.
Assuntos
Cobre , Luciferases de Vaga-Lume , Cobre/farmacologia , Luciferases/genética , Oxirredução , Luciferases de Vaga-Lume/metabolismo , Luciferina de Vaga-Lumes , Medições Luminescentes/métodos , LuminescênciaRESUMO
This work reports a new ATP-independent bioluminescent probe (bor-DTZ) for detecting hydrogen peroxide that is compatible with the Nanoluciferase enzyme. The probe is designed with an arylboronate ester protecting group appended to a diphenylterazine core via a self-immolative phenolate linker. Reaction with hydrogen peroxide reveals diphenylterazine, which can then react with Nanoluciferase to produce a detectable bioluminescent signal. Bor-DTZ shows a dose-dependent response to hydrogen peroxide and selectivity over other biologically relevant reactive oxygen species and can be applied to detect either intra- or extracellular species. We further demonstrate the ability of this platform to monitor fluxes in extracellular hydrogen peroxide in a breast cancer cell line in response to the anticancer treatment, cisplatin.
Assuntos
Trifosfato de Adenosina , Peróxido de Hidrogênio , Corantes Fluorescentes , Humanos , Peróxido de Hidrogênio/metabolismo , Células MCF-7 , Espécies Reativas de OxigênioRESUMO
The connecting peptide (C-peptide) is a hormone with promising health benefits in ameliorating diabetes-related complications, yet mechanisms remain elusive. Emerging studies point to a possible dependence of peptide activity on bioavailable metals, particularly Cu(II) and Zn(II). However, little is known about the chemical nature of the interactions, hindering advances in its therapeutic applications. This work uncovers the Cu(II)-binding site in C-peptide that may be key to understanding its metal-dependent function. A combination of spectroscopic studies reveal that Cu(II) and Zn(II) bind to C-peptide at specific residues in the N-terminal region of the peptide and that Cu(II) is able to displace Zn(II) for C-peptide binding. The data point to a Cu(II)-binding site consisting of 1N3O square-planar coordination that is entropically driven. Furthermore, the entire random coil peptide sequence is needed for specific metal binding as mutations and truncations reshuffle the coordinating residues. These results expand our understanding of how metals influence hormone activity and facilitate the discovery and validation of both new and established paradigms in peptide biology.
Assuntos
Peptídeo C/metabolismo , Cobre/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Peptídeo C/química , Ligação Proteica , Conformação Proteica , Termodinâmica , Zinco/metabolismoRESUMO
Iron is an essential metal for all organisms, yet disruption of its homeostasis, particularly in labile forms that can contribute to oxidative stress, is connected to diseases ranging from infection to cancer to neurodegeneration. Iron deficiency is also among the most common nutritional deficiencies worldwide. To advance studies of iron in healthy and disease states, we now report the synthesis and characterization of iron-caged luciferin-1 (ICL-1), a bioluminescent probe that enables longitudinal monitoring of labile iron pools (LIPs) in living animals. ICL-1 utilizes a bioinspired endoperoxide trigger to release d-aminoluciferin for selective reactivity-based detection of Fe2+ with metal and oxidation state specificity. The probe can detect physiological changes in labile Fe2+ levels in live cells and mice experiencing iron deficiency or overload. Application of ICL-1 in a model of systemic bacterial infection reveals increased iron accumulation in infected tissues that accompany transcriptional changes consistent with elevations in both iron acquisition and retention. The ability to assess iron status in living animals provides a powerful technology for studying the contributions of iron metabolism to physiology and pathology.
Assuntos
Infecções por Acinetobacter/metabolismo , Anemia Ferropriva/metabolismo , Luciferina de Vaga-Lumes/análise , Corantes Fluorescentes/análise , Sobrecarga de Ferro/metabolismo , Ferro/metabolismo , 2,2'-Dipiridil/farmacologia , Infecções por Acinetobacter/genética , Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/patologia , Acinetobacter baumannii/patogenicidade , Acinetobacter baumannii/fisiologia , Anemia Ferropriva/genética , Anemia Ferropriva/patologia , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Cátions Bivalentes , Modelos Animais de Doenças , Compostos Férricos/farmacologia , Luciferina de Vaga-Lumes/análogos & derivados , Luciferina de Vaga-Lumes/síntese química , Corantes Fluorescentes/síntese química , Regulação da Expressão Gênica , Hepcidinas/genética , Hepcidinas/metabolismo , Homeostase/genética , Sobrecarga de Ferro/genética , Sobrecarga de Ferro/patologia , Proteína 1 Reguladora do Ferro/genética , Proteína 1 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/metabolismo , Medições Luminescentes , Camundongos , Camundongos Transgênicos , Compostos de Amônio Quaternário/farmacologia , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Transdução de Sinais , Transferrina/genética , Transferrina/metabolismoRESUMO
Acinetobacter baumannii is a Gram-negative opportunistic pathogen that causes diverse infections, including pneumonia, bacteremia, and wound infections. Due to multiple intrinsic and acquired antimicrobial-resistance mechanisms, A. baumannii isolates are commonly multidrug resistant, and infections are notoriously difficult to treat. The World Health Organization recently highlighted carbapenem-resistant A. baumannii as a "critical priority" for the development of new antimicrobials because of the risk to human health posed by this organism. Therefore, it is important to discover the mechanisms used by A. baumannii to survive stresses encountered during infection in order to identify new drug targets. In this study, by use of in vivo imaging, we identified hydrogen peroxide (H2O2) as a stressor produced in the lung during A. baumannii infection and defined OxyR as a transcriptional regulator of the H2O2 stress response. Upon exposure to H2O2, A. baumannii differentially transcribes several hundred genes. However, the transcriptional upregulation of genes predicted to detoxify hydrogen peroxide is abolished in an A. baumannii strain in which the transcriptional regulator oxyR is genetically inactivated. Moreover, inactivation of oxyR in both antimicrobial-susceptible and multidrug-resistant A. baumannii strains impairs growth in the presence of H2O2 OxyR is a direct regulator of katE and ahpF1, which encode the major H2O2-degrading enzymes in A. baumannii, as confirmed through measurement of promoter binding by recombinant OxyR in electromobility shift assays. Finally, an oxyR mutant is less fit than wild-type A. baumannii during infection of the murine lung. This work reveals a mechanism used by this important human pathogen to survive H2O2 stress encountered during infection.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Anti-Infecciosos Locais/metabolismo , Regulação Bacteriana da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Oxidantes/metabolismo , Proteínas Repressoras/metabolismo , Estresse Fisiológico , Infecções por Acinetobacter/imunologia , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/genética , Acinetobacter baumannii/fisiologia , Animais , CamundongosRESUMO
The connecting peptide (C-peptide) has received increased attention for its potential therapeutic effects in ameliorating illnesses such as kidney disease and diabetes. Although the mechanism of C-peptide signaling remains elusive, evidence supports its internalization and intracellular function. Emerging research is uncovering the diverse biological roles metals play in controlling and affecting the function of bioactive peptides. The work presented herein investigates interactions between C-peptide and first-row d-block transition metals, as well as their effects on C-peptide internalization into cells. Through spectroscopic techniques, it is demonstrated that CrIII , CuII , and ZnII bind to C-peptide with differing stoichiometries and biologically relevant affinities. In addition, metal binding elicits both subtle changes in secondary structure and inhibits adoption of an α-helical character in environments where the dielectric constants are reduced. This study shows how metal ions can modulate peptide hormone activity through subtle structural changes to disrupt cellular uptake.
Assuntos
Peptídeo C/química , Peptídeo C/metabolismo , Quelantes/farmacologia , Metais/farmacologia , Peptídeo C/efeitos dos fármacos , Células HEK293 , Humanos , Ligação ProteicaRESUMO
Copper is a required metal nutrient for life, but global or local alterations in its homeostasis are linked to diseases spanning genetic and metabolic disorders to cancer and neurodegeneration. Technologies that enable longitudinal in vivo monitoring of dynamic copper pools can help meet the need to study the complex interplay between copper status, health, and disease in the same living organism over time. Here, we present the synthesis, characterization, and in vivo imaging applications of Copper-Caged Luciferin-1 (CCL-1), a bioluminescent reporter for tissue-specific copper visualization in living animals. CCL-1 uses a selective copper(I)-dependent oxidative cleavage reaction to release d-luciferin for subsequent bioluminescent reaction with firefly luciferase. The probe can detect physiological changes in labile Cu+ levels in live cells and mice under situations of copper deficiency or overload. Application of CCL-1 to mice with liver-specific luciferase expression in a diet-induced model of nonalcoholic fatty liver disease reveals onset of hepatic copper deficiency and altered expression levels of central copper trafficking proteins that accompany symptoms of glucose intolerance and weight gain. The data connect copper dysregulation to metabolic liver disease and provide a starting point for expanding the toolbox of reactivity-based chemical reporters for cell- and tissue-specific in vivo imaging.
Assuntos
Cobre/deficiência , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Luciferina de Vaga-Lumes , Substâncias Luminescentes , Medições Luminescentes/métodos , Masculino , Metalochaperonas/metabolismo , Camundongos , Camundongos Transgênicos , Imagem Molecular/métodos , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Hepatopatia Gordurosa não Alcoólica/etiologiaRESUMO
Copper deficiency is implicated in a variety of genetic, neurological, cardiovascular, and metabolic diseases. Current approaches for addressing copper deficiency rely on generic copper supplementation, which can potentially lead to detrimental off-target metal accumulation in unwanted tissues and subsequently trigger oxidative stress and damage cascades. Here we present a new modular platform for delivering metal ions in a tissue-specific manner and demonstrate liver-targeted copper supplementation as a proof of concept of this strategy. Specifically, we designed and synthesized an N-acetylgalactosamine-functionalized ionophore, Gal-Cu(gtsm), to serve as a copper-carrying "Trojan Horse" that targets liver-localized asialoglycoprotein receptors (ASGPRs) and releases copper only after being taken up by cells, where the reducing intracellular environment triggers copper release from the ionophore. We utilized a combination of bioluminescence imaging and inductively coupled plasma mass spectrometry assays to establish ASGPR-dependent copper accumulation with this reagent in both liver cell culture and mouse models with minimal toxicity. The modular nature of our synthetic approach presages that this platform can be expanded to deliver a broader range of metals to specific cells, tissues, and organs in a more directed manner to treat metal deficiency in disease.
Assuntos
Acetilgalactosamina/metabolismo , Cobre/administração & dosagem , Cobre/farmacocinética , Suplementos Nutricionais , Portadores de Fármacos/metabolismo , Ionóforos/metabolismo , Fígado/metabolismo , Acetilgalactosamina/síntese química , Acetilgalactosamina/química , Animais , Receptor de Asialoglicoproteína/metabolismo , Suplementos Nutricionais/análise , Portadores de Fármacos/síntese química , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Ionóforos/síntese química , Ionóforos/química , CamundongosRESUMO
Cobalt(III) Schiff base complexes ([Co(acacen)(L)2](+), where L = NH3) inhibit histidine-containing proteins through dissociative exchange of the labile axial ligands (L). This work investigates axial ligand exchange dynamics of [Co(acacen)(L)2](+) complexes toward the development of protein inhibitors that are activated by external triggers such as light irradiation. We sought to investigate ligand exchange dynamics to design a Co(III) complex that is substitutionally inert under normal physiological conditions for selective activation. Fluorescent imidazoles (C3Im) were prepared as axial ligands in [Co(acacen)(L)2](+) to produce complexes (CoC3Im) that could report on ligand exchange and, thus, complex stability. These fluorescent imidazole reporters guided the design of a new dinuclear Co(III) Schiff base complex containing bridging diimidazole ligands, which exhibits enhanced stability to ligand exchange with competing imidazoles and to hydrolysis within a biologically relevant pH range. These studies inform the design of biocompatible Co(III) Schiff base complexes that can be selectively activated for protein inhibition with spatial and temporal specificity.
Assuntos
Cobalto , Complexos de Coordenação/farmacologia , Corantes Fluorescentes/farmacologia , Oligopeptídeos/antagonistas & inibidores , Bases de Schiff/farmacologia , Complexos de Coordenação/síntese química , Corantes Fluorescentes/síntese química , Imidazóis/síntese química , Ligantes , Bases de Schiff/síntese químicaRESUMO
Oligomers of the Aß42 peptide are significant neurotoxins linked to Alzheimer's disease (AD). Histidine (His) residues present at the N terminus of Aß42 are believed to influence toxicity by either serving as metal-ion binding sites (which promote oligomerization and oxidative damage) or facilitating synaptic binding. Transition metal complexes that bind to these residues and modulate Aß toxicity have emerged as therapeutic candidates. Cobalt(III) Schiff base complexes (Co-sb) were evaluated for their ability to interact with Aß peptides. HPLC-MS, NMR, fluorescence, and DFT studies demonstrated that Co-sb complexes could interact with the His residues in a truncated Aß16 peptide representing the Aß42 N terminus. Coordination of Co-sb complexes altered the structure of Aß42 peptides and promoted the formation of large soluble oligomers. Interestingly, this structural perturbation of Aß correlated to reduced synaptic binding to hippocampal neurons. These results demonstrate the promise of Co-sb complexes in anti-AD therapeutic approaches.
Assuntos
Peptídeos beta-Amiloides/química , Cobalto/química , Histidina/química , Compostos Organometálicos/química , Conformação Molecular , Bases de Schiff/químicaRESUMO
Transcription factors are key regulators in both normal and pathological cell processes. Affecting the activity of these proteins is a promising strategy for understanding gene regulation and developing effective therapeutics. Co(III) Schiff base complexes ([Co(acacen)(L)2](+) where L=labile axial ligands) have been shown to be potent inhibitors of a number of zinc metalloproteins including Cys2His2 zinc finger transcription factors. Inhibition by [Co(acacen)(L)2](+) of the target protein is believed to occur through a dissociative exchange of the labile axial ligands for histidine (His) residues essential for function. Here, we report a series of spectroscopic investigations with model peptides of zinc fingers that elucidate the interaction between [Co(acacen)(L)2](+) complexes and zinc finger transcription factors. Observed changes in NMR chemical shifts and 2D (1)H-(1)H NOESY NMR spectra demonstrate the preference of [Co(acacen)(L)2](+) complexes to coordinate His residues over other amino acids. The conformation of [Co(acacen)(L)2](+) upon His coordination was characterized by (1)H NMR spectroscopy, near-UV CD, and electronic absorption. These studies reveal that the resulting His-coordinated [Co(acacen)(L)2](+) complex possesses an octahedral structure. The effects of [Co(acacen)(L)2](+) complexes on the zinc-finger structure were assessed by the degree of hydrogen bonding (probed by 2D NMR spectroscopy) and secondary-structure profiles measured by far-UV CD. These structural studies demonstrate the ability of [Co(acacen)(L)2](+) complexes to disrupt the ßßα structure of zinc fingers, resulting in primarily random-coil conformations. A mechanism is described wherein [Co(acacen)(L)2](+) complexes inhibit zinc finger transcription factor activity through selectively coordinating His residues in the zinc finger by dissociative ligand exchange and disrupting the ßßα structural motif required for gene regulation.
Assuntos
Cobalto/química , Cobalto/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Cistina/química , Histidina/química , Metaloproteínas/química , Oligopeptídeos/química , Bases de Schiff/química , Bases de Schiff/farmacologia , Fatores de Transcrição/química , Sítios de Ligação , Ligantes , Espectroscopia de Ressonância Magnética , Estrutura Secundária de Proteína , Dedos de ZincoRESUMO
We report the structural optimization and mechanistic investigation of a series of bioactivated magnetic resonance imaging contrast agents that transform from low relaxivity to high relaxivity in the presence of Zn(II). The change in relaxivity results from a structural transformation of the complex that alters the coordination environment about the Gd(III) center. Here, we have performed a series of systematic modifications to determine the structure that provides the optimal change in relaxivity in response to the presence of Zn(II). Relaxivity measurements in the presence and absence of Zn(II) were used in conjunction with measurements regarding water access (namely, number of water molecules bound) to the Gd(III) center and temperature-dependent (13)C NMR spectroscopy to determine how the coordination environment about the Gd(III) center is affected by the distance between the Zn(II)-binding domain and the Gd(III) chelate, the number of functional groups on the Zn(II)-binding domain, and the presence of Zn(II). The results of this study provide valuable insight into the design principles for future bioactivated magnetic resonance probes.
Assuntos
Meios de Contraste/química , Gadolínio/química , Imageamento por Ressonância Magnética/métodos , Zinco/química , Quelantes/química , Técnicas de Química Sintética , Espectroscopia de Ressonância Magnética/métodos , Relação Estrutura-Atividade , TemperaturaRESUMO
The kinetic and thermodynamic ligand exchange dynamics are important considerations in the rational design of metal-based therapeutics and therefore, require detailed investigation. Co(III) Schiff base complex derivatives of bis(acetylacetone)ethylenediimine [acacen] have been found to be potent enzyme and transcription factor inhibitors. These complexes undergo solution exchange of labile axial ligands. Upon dissociation, Co(III) irreversibly interacts with specific histidine residues of a protein, and consequently alters structure and causes inhibition. To guide the rational design of next generation agents, understanding the mechanism and dynamics of the ligand exchange process is essential. To investigate the lability, pH stability, and axial ligand exchange of these complexes in the absence of proteins, the pD- and temperature-dependent axial ligand substitution dynamics of a series of N-heterocyclic [Co(acacen)(X)(2)](+) complexes [where X = 2-methylimidazole (2MeIm), 4-methylimidazole (4MeIm), ammine (NH(3)), N-methylimidazole (NMeIm), and pyridine (Py)] were characterized by NMR spectroscopy. The pD stability was shown to be closely related to the nature of the axial ligand with the following trend toward aquation: 2MeIm > NH(3) â« 4MeIm > Py > Im > NMeIm. Reaction of each [Co(III)(acacen)(X)(2)](+) derivative with 4MeIm showed formation of a mixed ligand Co(III) intermediate via a dissociative ligand exchange mechanism. The stability of the mixed ligand adduct was directly correlated to the pD-dependent stability of the starting Co(III) Schiff base with respect to [Co(acacen)(4MeIm)(2)](+). Crystal structure analysis of the [Co(acacen)(X)(2)](+) derivatives confirmed the trends in stability observed by NMR spectroscopy. Bond distances between the Co(III) and the axial nitrogen atoms were longest in the 2MeIm derivative as a result of distortion in the planar tetradentate ligand, and this was directly correlated to axial ligand lability and propensity toward exchange.
Assuntos
Cobalto/química , Complexos de Coordenação/química , Bases de Schiff/química , Complexos de Coordenação/classificação , Cristalografia por Raios X , Estabilidade de Medicamentos , Concentração de Íons de Hidrogênio , Ligantes , Espectroscopia de Ressonância Magnética , Estrutura MolecularRESUMO
Oxytocin is a 9-amino acid peptide hormone. Since its discovery in 1954, it has most commonly been studied in relation to its role in stimulating parturition and lactation. However, it is now known that oxytocin has a widely diverse set of functions throughout the body including neuromodulation, bone growth, and inflammation. Previous research has suggested that divalent metal ions may be required for oxytocin activity, but the exact metal species and specific pathways have yet to be fully elucidated. In this work, we focus on characterizing copper and zinc bound forms of oxytocin and related analogs through far-UV circular dichroism. We report that Cu(ii) and Zn(ii) bind uniquely to oxytocin and all analogs investigated. Furthermore, we investigate how these metal bound forms may affect downstream signaling of MAPK activation upon receptor binding. We find that both Cu(ii) and Zn(ii) bound oxytocin attenuates the activation of the MAPK pathway upon receptor binding relative to oxytocin alone. Interestingly, we observed that Zn(ii) bound forms of linear oxytocin facilitate increased MAPK signaling. This study lays the foundation for future work on elucidating the metal effects on oxytocin's diverse bioactivity.
RESUMO
Background and aims: Major clinical manifestations of Wilson disease (WD) are related to copper accumulation in the liver and the brain, and little is known about other tissues involvement in metabolic changes in WD. In vitro studies suggested that the loss of intestinal ATP7B could contribute to metabolic dysregulation in WD. We tested this hypothesis by evaluating gut microbiota and lipidome in two mouse models of WD and by characterizing a new mouse model with a targeted deletion of Atp7b in intestine. Methods: Cecal content 16S sequencing and untargeted hepatic and plasma lipidome analyses in the Jackson Laboratory toxic-milk and the Atp7b null global knockout mouse models of WD were profiled and integrated. Intestine-specific Atp7b knockout mice ( Atp7b ΔIEC ) was generated using B6.Cg-Tg(Vil1-cre)997Gum/J mice and Atp7b Lox/Lox mice, and characterized using targeted lipidome analysis following a high-fat diet challenge. Results: Gut microbiota diversity was reduced in animal models of WD. Comparative prediction analysis revealed amino acid, carbohydrate, and lipid metabolism functions to be dysregulated in the WD gut microbial metagenome. Liver and plasma lipidomic profiles showed dysregulated tri- and diglyceride, phospholipid, and sphingolipid metabolism in WD models. When challenged with a high-fat diet, Atp7b ΔIEC mice exhibited profound alterations to fatty acid desaturation and sphingolipid metabolism pathways as well as altered APOB48 distribution in intestinal epithelial cells. Conclusion: Coordinated changes of gut microbiome and lipidome analyses underlie systemic metabolic manifestations in murine WD. Intestine-specific ATP7B deficiency affected both intestinal and systemic response to a high-fat challenge. WD is a systemic disease in which intestinal-specific ATP7B loss and diet influence phenotypic presentations.
RESUMO
BACKGROUND: The clinical manifestations of Wilson disease (WD) are related to copper accumulation in the liver and the brain, but little is known about other tissue involvement regarding metabolic changes in WD. In vitro studies suggested that the loss of intestinal ATP7B affects metabolic dysregulation in WD. We tested this hypothesis by evaluating the gut microbiota and lipidome in 2 mouse models of WD and by characterizing a new mouse model with a targeted deletion of Atp7b in the intestine. METHODS: Cecal content 16S sequencing and untargeted hepatic and plasma lipidome analyses in the Jackson Laboratory toxic-milk and the Atp7b null global knockout mouse models of WD were profiled and integrated. Intestine-specific Atp7b knockout mice (Atp7bΔIEC) were generated and characterized using targeted lipidome analysis following a high-fat diet challenge. RESULTS: Gut microbiota diversity was reduced in animal models of WD. Comparative prediction analysis revealed amino acid, carbohydrate, and lipid metabolism functions to be dysregulated in the WD gut microbial metagenome. Liver and plasma lipidomic profiles showed dysregulated triglyceride and diglyceride, phospholipid, and sphingolipid metabolism in WD models. However, Atp7bΔIEC mice did not show gut microbiome differences compared to wild type. When challenged with a high-fat diet, Atp7bΔIEC mice exhibited profound alterations to fatty acid desaturation and sphingolipid metabolism pathways as well as altered APOB48 distribution in intestinal epithelial cells. CONCLUSIONS: Gut microbiome and lipidome underlie systemic metabolic manifestations in murine WD. Intestine-specific ATP7B deficiency affected both intestinal and systemic response to a high-fat challenge but not the microbiome profile, at least at early stages. WD is a systemic disease in which intestinal-specific ATP7B loss and diet influence the phenotype and the lipidome profile.