RESUMO
Desferrioxamine (DFO) has long been considered the gold standard chelator for incorporating [89Zr]Zr4+ in radiopharmaceuticals for positron emission tomography (PET) imaging. To improve the stability of DFO with zirconium-89 and to expand its coordination sphere to enable binding of large therapeutic radiometals, we have synthesized the highest denticity DFO derivatives to date: dodecadentate DFO2 and DFO2p. In this study, we describe the synthesis and characterization of a novel DFO-based chelator, DFO2p, which is comprised of two DFO strands connected by an p-NO2-phenyl linker and therefore contains double the chelating moieties of DFO (potential coordination number up to 12 vs 6). The chelator DFO2p offers an optimized synthesis comprised of only a single reaction step and improves water solubility relative to DFO2, but the shorter linker reduces molecular flexibility. Both DFO2 and DFO2p, each with 6 potential hydroxamate ligands, are able to reach a more energetically favorable 8-coordinate environment for Zr(IV) than DFO. The zirconium(IV) coordination environment of these complexes were evaluated by a combination of density functional theory (DFT) calculations and synchrotron spectroscopy (extended X-ray absorption fine structure), which suggest the inner-coordination sphere of zirconium(IV) to be comprised of the outermost four hydroxamate ligands. These results also confirm a single Zr(IV) in each chelator, and the hydroxide ligands which complete the coordination sphere of Zr(IV)-DFO are absent from Zr(IV)-DFO2 and Zr(IV)-DFO2p. Radiochemical stability studies with zirconium-89 revealed the order of real-world stability to be DFO2 > DFO2p â« DFO. The zirconium-89 complexes of these new high-denticity chelators were found to be far more stable than DFO, and the decreased molecular flexibility of DFO2p, relative to DFO2, could explain its decreased stability, relative to DFO2.
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Although Alzheimer's disease (AD) was first described over a century ago, it remains the leading cause of age-related dementia. Innumerable changes have been linked to the pathology of AD; however, there remains much discord regarding which might be the initial cause of the disease. The "amyloid cascade hypothesis" proposes that the amyloid ß (Aß) peptide is central to disease pathology, which is supported by elevated Aß levels in the brain before the development of symptoms and correlations of amyloid burden with cognitive impairment. The "metals hypothesis" proposes a role for metal ions such as iron, copper, and zinc in the pathology of AD, which is supported by the accumulation of these metals within amyloid plaques in the brain. Metals have been shown to induce aggregation of Aß, and metal ion chelators have been shown to reverse this reaction in vitro. 8-Hydroxyquinoline-based chelators showed early promise as anti-Alzheimer's drugs. Both 5-chloro-7-iodo-8-hydroxyquinoline (CQ) and 5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline (PBT2) underwent unsuccessful clinical trials for the treatment of AD. To gain insight into the mechanism of action of 8HQs, we have investigated the potential interaction of CQ, PBT2, and 5,7-dibromo-8-hydroxyquinoline (B2Q) with Cu(II)-bound Aß(1-42) using X-ray absorption spectroscopy (XAS), high energy resolution fluorescence detected (HERFD) XAS, and electron paramagnetic resonance (EPR). By XAS, we found CQ and B2Q sequestered â¼83% of the Cu(II) from Aß(1-42), whereas PBT2 sequestered only â¼59% of the Cu(II) from Aß(1-42), suggesting that CQ and B2Q have a higher relative Cu(II) affinity than PBT2. From our EPR, it became clear that PBT2 sequestered Cu(II) from a heterogeneous mixture of Cu(II)Aß(1-42) species in solution, leaving a single Cu(II)Aß(1-42) species. It follows that the Cu(II) site in this Cu(II)Aß(1-42) species is inaccessible to PBT2 and may be less solvent-exposed than in other Cu(II)Aß(1-42) species. We found no evidence to suggest that these 8HQs form ternary complexes with Cu(II)Aß(1-42).
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Clioquinol , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/química , Quelantes/farmacologia , Quelantes/uso terapêutico , Clioquinol/análogos & derivados , Clioquinol/química , Cobre/química , Humanos , Íons , Metais , Oxiquinolina/química , Oxiquinolina/farmacologia , Fragmentos de Peptídeos , Solventes , ZincoRESUMO
PBT2 (5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline) is a small Cu(II)-binding drug that has been investigated in the treatment of neurodegenerative diseases, namely, Alzheimer's disease (AD). PBT2 is thought to be highly effective at crossing the blood-brain barrier and has been proposed to exert anti-Alzheimer's effects through the modulation of metal ion concentrations in the brain, specifically the sequestration of Cu(II) from amyloid plaques. However, despite promising initial results in animal models and in clinical trials where PBT2 was shown to improve cognitive function, larger-scale clinical trials did not find PBT2 to have a significant effect on the amyloid plaque burden compared with controls. We propose that the results of these clinical trials likely point to a more complex mechanism of action for PBT2 other than simple Cu(II) sequestration. To this end, herein we have investigated the solution chemistry of Cu(II) coordination by PBT2 primarily using X-ray absorption spectroscopy (XAS), high-energy-resolution fluorescence-detected XAS, and electron paramagnetic resonance. We propose that a novel bis-PBT2 Cu(II) complex with asymmetric coordination may coexist in solution with a symmetric four-coordinate Cu(II)-bis-PBT2 complex distorted from coplanarity. Additionally, PBT2 is a more flexible ligand than other 8HQs because it can act as both a bidentate and a tridentate ligand as well as coordinate Cu(II) in both 1:1 and 2:1 PBT2/Cu(II) complexes.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Quelantes/uso terapêutico , Clioquinol/análogos & derivados , Complexos de Coordenação/uso terapêutico , Cobre/uso terapêutico , Fármacos Neuroprotetores/farmacologia , Deficiências na Proteostase/tratamento farmacológico , Animais , Quelantes/síntese química , Quelantes/química , Clioquinol/química , Clioquinol/uso terapêutico , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Cobre/química , Teoria da Densidade Funcional , Humanos , Ligantes , Estrutura Molecular , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Espectroscopia por Absorção de Raios XRESUMO
Positron emission tomography (PET) using radiolabeled, monoclonal antibodies has become an effective, noninvasive method for tumor detection and is a critical component of targeted radionuclide therapy. Metal ion chelator and bacterial siderophore desferrioxamine (DFO) is the gold standard compound for incorporation of zirconium-89 in radiotracers for PET imaging because it is thought to form a stable chelate with [89Zr]Zr4+. However, DFO may not bind zirconium-89 tightly in vivo, with free zirconium-89 reportedly liberated into the bones of experimental mouse models. Although high bone uptake has not been observed to date in humans, this potential instability has been proposed to be related to the unsaturated coordination sphere of [89Zr]Zr-DFO, which is thought to consist of the 3 hydroxamate groups of DFO and 1 or 2 water molecules. In this study, we have used a combination of X-ray absorption spectroscopy and density functional theory (DFT) geometry optimization calculations to further probe the coordination chemistry of this complex in solution. We find the extended X-ray absorption fine structure (EXAFS) curve fitting of an aqueous solution of Zr(IV)-DFO to be consistent with an 8-coordinate Zr with oxygen ligands. DFT calculations suggest that the most energetically favorable Zr(IV) coordination environment in DFO likely consists of the 3 hydroxamate ligands from DFO, each with bidentate coordination, and 2 hydroxide ligands. Further EXAFS curve fitting provides additional support for this model. Therefore, we propose that the coordination sphere of Zr(IV)-DFO is most likely completed by 2 hydroxide ligands rather than 2 water molecules, forming Zr(DFO)(OH)2.
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8-Hydroxyquinolines (8HQs) are a family of lipophilic metal ion chelators that have been used in a range of analytical and pharmaceutical applications over the last 100 years. More recently, CQ (clioquinol; 5-chloro-7-iodo-8-hydroxyquinoline) and PBT2 (5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline) have undergone clinical trials for the treatment of Alzheimer's disease and Huntington's disease. Because CQ and PBT2 appear to redistribute metals into cells, these compounds have been redefined as copper and zinc ionophores. Despite the attention surrounding the clinical trials and the clear link between 8HQs and metals, the fundamental solution chemistry of how these compounds bind divalent metals such as copper and zinc, as well as their mechanism(s) of action in mammalian systems, remains poorly understood. In this study, we used a combination of X-ray absorption spectroscopy (XAS), high-energy resolution fluorescence detected (HERFD) XAS, electron paramagnetic resonance (EPR), and UV-visible absorption spectroscopies to investigate the aqueous solution chemistry of a range of 8HQ derivatives. To circumvent the known solubility issues with 8HQ compounds and their complexes with Cu(II), and to avoid the use of abiological organic solvents, we have devised a surfactant buffer system to investigate these Cu(II) complexes in aqueous solution. Our study comprises the first comprehensive investigation of the Cu(II) complexes formed with many 8HQs of interest in aqueous solution, and it provides the first structural information on some of these complexes. We find that halogen substitutions in 8HQ derivatives appear to have little effect on the Cu(II) coordination environment; 5,7-dihalogenated 8HQ conformers all have a pseudo square planar Cu(II) bound by two quinolin-8-olate anions, in agreement with previous studies. Conversely, substituents in the 2-position of the 8HQ moiety appear to cause significant distortions from the typical square-planar-like coordination of most Cu(II)-bis-8HQ complexes, such that the 8HQ moieties in the Cu(II)-bis-8HQ complex are rotated approximately 30-40° apart in a "propeller-like" arrangement.
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Pathogenic lymphocytes aberrantly recognize and mount an immune response against self-antigens, leading to the destruction of healthy cells, tissues and organs. Recent studies have shown that both B and T lymphocytes contribute to the development, prevention and modulation of various autoimmune diseases. Regulatory T and B cell subsets appear to play a prominent role in the prevention of autoimmune diseases. The recent identification of novel regulatory Th17 cells, termed as Treg17 cells, has expanded the scope of regulatory T lymphocytes (Treg cells) in the prevention of autoimmune diseases. Similarly, novel regulatory B cell subsets, termed as Breg cells, acting on their own or by inducing Treg cells have extended the role of B lymphocytes in the prevention and regulation of autoimmune diseases. We suggest that Treg17 cells and Breg cells have an important immunoregulatory role in autoimmune diseases.
Assuntos
Doenças Autoimunes/imunologia , Linfócitos B Reguladores/imunologia , Linfócitos B/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , HumanosRESUMO
Stress is known to impede certain host defense mechanisms, including those governed by conventional T lymphocytes. However, whether innate-like T lymphocytes, such as invariant natural killer T (iNKT) and mucosa-associated invariant T (MAIT) cells, are impacted by stress is unclear. Herein, we report that prolonged psychological stress caused by physical confinement results in robust upregulation of T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), an immune checkpoint receptor that controls antitumor and antiviral immune responses. Elevated TIGIT expression was found not only on NK and conventional T cells, but also on iNKT and MAIT cells. Stress-provoked TIGIT upregulation was reversed through treatment with the glucocorticoid receptor (GR) antagonist RU486, but not with 6-hydroxydopamine that induces chemical sympathectomy. A Cre/Lox gene targeting model in which GR was ablated in cells expressing Lck under its proximal promoter revealed that TIGIT upregulation in stressed animals stems from direct GR signaling in T and iNKT cells. In fact, long-term oral administration of exogenous corticosterone (CS) to wild-type C57BL/6 (B6) mice was sufficient to increase TIGIT expression levels on T and iNKT cells. In vitro treatment with CS also potently and selectively upregulated TIGIT, but not CTLA-4 or LAG-3, on mouse iNKT and MAIT hybridomas. These results were recapitulated using primary hepatic iNKT and MAIT cells from wild-type B6 and B6.MAITCAST mice, respectively. Subjecting B6.MAITCAST mice to physical restraint also raised the frequency of TIGIT+ cells among hepatic MAIT cells in a GR-dependent manner. Finally, we found that TIGIT is similarly upregulated in a chronic variable stress model in which animals are exposed to unpredictable heterotypic stressors without developing habituation. Taken together, our findings link, for the first time to our knowledge, GR signaling to TIGIT expression. We propose that glucocorticoid hormones dampen immune responses, in part, by enhancing TIGIT expression across multiple critical subsets of effector lymphocytes, including innate-like T cells. Therefore, TIGIT may constitute an attractive target in immune-enhancing interventions for sustained physiological stress.
Assuntos
Células T Invariantes Associadas à Mucosa/metabolismo , Células T Matadoras Naturais/metabolismo , Receptores Imunológicos/metabolismo , Estresse Psicológico/metabolismo , Animais , Feminino , Ativação Linfocitária , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células T Invariantes Associadas à Mucosa/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Glucocorticoides/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Transdução de Sinais , Estresse Psicológico/imunologia , Ativação Transcricional , Regulação para CimaRESUMO
[NiFe]-hydrogenase enzymes catalyze the reversible oxidation of hydrogen at a bimetallic cluster and are used by bacteria and archaea for anaerobic growth and pathogenesis. Maturation of the [NiFe]-hydrogenase requires several accessory proteins to assemble and insert the components of the active site. The penultimate maturation step is the delivery of nickel to a primed hydrogenase enzyme precursor protein, a process that is accomplished by two nickel metallochaperones, the accessory protein HypA and the GTPase HypB. Recent work demonstrated that nickel is rapidly transferred to HypA from GDP-loaded HypB within the context of a protein complex in a nickel selective and unidirectional process. To investigate the mechanism of metal transfer, we examined the allosteric effects of nucleotide cofactors and partner proteins on the nickel environments of HypA and HypB by using a combination of biochemical, microbiological, computational, and spectroscopic techniques. We observed that loading HypB with either GDP or a nonhydrolyzable GTP analogue resulted in a similar nickel environment. In addition, interaction with a mutant version of HypA with disrupted nickel binding, H2Q-HypA, does not induce substantial changes to the HypB G-domain nickel site. Instead, the results demonstrate that HypB modifies the acceptor site of HypA. Analysis of a peptide maquette derived from the N-terminus of HypA revealed that nickel is predominately coordinated by atoms from the N-terminal Met-His motif. Furthermore, HypA is capable of two nickel-binding modes at the N-terminus, a HypB-induced mode and a binding mode that mirrors the peptide maquette. Collectively, these results reveal that HypB brings about changes in the nickel coordination of HypA, providing a mechanism for the HypB-dependent control of the acquisition and release of nickel by HypA.
Assuntos
Proteínas de Transporte/química , Complexos de Coordenação/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Níquel/química , Proteínas de Transporte/metabolismo , Complexos de Coordenação/metabolismo , Proteínas de Escherichia coli/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Moleculares , Níquel/metabolismoRESUMO
Alzheimer's disease (AD) is the main cause of age-related dementia and currently affects approximately 5.7 million Americans. Major brain changes associated with AD pathology include accumulation of amyloid beta (Aß) protein fragments and formation of extracellular amyloid plaques. Redox-active metals mediate oligomerization of Aß, and the resultant metal-bound oligomers have been implicated in the putative formation of harmful, reactive species that could contribute to observed oxidative damage. In isolated plaque cores, Cu(II) is bound to Aß via histidine residues. Despite numerous structural studies of Cu(II) binding to synthetic Aß in vitro, there is still uncertainty surrounding Cu(II) coordination in Aß. In this study, we used X-ray absorption spectroscopy (XAS) and high energy resolution fluorescence detected (HERFD) XAS to investigate Cu(II) coordination in Aß(1-42) under various solution conditions. We found that the average coordination environment in Cu(II)Aß(1-42) is sensitive to X-ray photoreduction, changes in buffer composition, peptide concentration, and solution pH. Fitting of the extended X-ray absorption fine structure (EXAFS) suggests Cu(II) is bound in a mixture of coordination environments in monomeric Aß(1-42) under all conditions studied. However, it was evident that on average only a single histidine residue coordinates Cu(II) in monomeric Aß(1-42) at pH 6.1, in addition to 3 other oxygen or nitrogen ligands. Cu(II) coordination in Aß(1-42) at pH 7.4 is similarly 4-coordinate with oxygen and nitrogen ligands, although an average of 2 histidine residues appear to coordinate at this pH. At pH 9.0, the average Cu(II) coordination environment in Aß(1-42) appears to be 5-coordinate with oxygen and nitrogen ligands, including two histidine residues.
Assuntos
Peptídeos beta-Amiloides/química , Cobre/química , Histidina/química , Fragmentos de Peptídeos/química , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Cobre/metabolismo , Histidina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Oxirredução , Fragmentos de Peptídeos/metabolismo , Espectroscopia por Absorção de Raios XRESUMO
Alzheimer's disease (AD) is a major international health and economic concern. A key pathological feature of AD is so-called "amyloid-ß-plaques", or "Aß-plaques", which are deposits of aggregated protein, enriched with the Aß fragment of amyloid precursor protein. Despite their name, the deposits are not pure Aß and have a heterogeneous, chemically complex composition that can include multiple proteins, lipids, and metal ions (Fe, Cu, or Zn). Despite extensive research, it is still uncertain whether Aß-plaques are a cause or a consequence of AD pathology. Further characterization of the elemental and biochemical composition within and surrounding Aß-plaques, and knowledge of how composition varies with disease state or progression, may provide important insight into the relationship between Aß-plaques and AD pathology. With this aim in mind, herein we demonstrate a multimodal spectroscopic imaging workflow to better characterize the complex composition of Aß-plaques. Our approach incorporates several spectroscopic imaging techniques, such as Fourier transform infrared spectroscopic imaging (FTIR), Raman microscopy, and X-ray fluorescence microscopy (XFM). While FTIR, Raman, and XFM have been used previously, mostly in isolation, to study Aß-plaques, application of all three techniques, in combination with histology and fluorescence microscopy, has not been reported previously. We demonstrate that a multimodal workflow, incorporating all three methods on adjacent or serial tissue sections, can reveal substantial complementary information about the biochemical and elemental composition of Aß-plaques. Information revealed by the method includes the relative content and distribution of aggregated protein, total lipid, lipid esters, cholesterol, and metals (Fe, Cu, or Zn).
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Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Metais/metabolismo , Agregação Patológica de Proteínas/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Animais , Camundongos , Camundongos Transgênicos , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia , Espectrometria por Raios X , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral RamanRESUMO
Copper is an essential nutrient required for many biological processes involved in primary metabolism, but free copper is toxic due to its ability to catalyze formation of free radicals. To prevent toxic effects, in the cell copper is bound to proteins and low molecular weight compounds, such as glutathione, at all times. The widely used chemotherapy agent cisplatin is known to bind to copper-transporting proteins, including copper chaperone Atox1. Cisplatin interactions with Atox1 and other copper transporters are linked to cancer resistance to platinum-based chemotherapy. Here we analyze the binding of copper and cisplatin to Atox1 in the presence of glutathione under redox conditions that mimic intracellular environment. We show that copper(I) and glutathione form large polymers with a molecular mass of approximately 8 kDa, which can transfer copper to Atox1. Cisplatin also can form polymers with glutathione, albeit at a slower rate. Analysis of simultaneous binding of copper and cisplatin to Atox1 under physiological conditions shows that both metals are bound to the protein through copper-sulfur-platinum bridges.
Assuntos
Cisplatino/metabolismo , Cobre/metabolismo , Glutationa/metabolismo , Metalochaperonas/metabolismo , Platina/metabolismo , Enxofre/metabolismo , Sítios de Ligação , Cisplatino/química , Cobre/química , Proteínas de Transporte de Cobre , Glutationa/química , Metalochaperonas/química , Metalochaperonas/isolamento & purificação , Chaperonas Moleculares , Conformação Molecular , Simulação de Dinâmica Molecular , Método de Monte Carlo , Oxirredução , Platina/química , Enxofre/químicaRESUMO
OBJECTIVE: Data from a small study testing imatinib to treat SSc were used to determine if cytokine changes were related to differences in clinical parameters to model future early phase trials pairing cytokine changes and clinical parameters. METHODS: Plasma and punch skin biopsy specimens collected at baseline and 6 months were analysed for levels of 26 fibrotic and inflammatory cytokines using multiplexed immunoassays and ELISA. Seven of nine patients on active treatment had paired data. Biopsies were biopulverized and standardized to protein levels in the tissue homogenate. Plasma was frozen at -80°C and analysed using multiplexed immunoassays or ELISAs standardized to CRP. Correlations between fold changes in cytokines and differences in clinical parameters (skin score, physician and patient global assessments and HAQ) were performed. P < 0.01 was considered significant. RESULTS: After 6 months of imatinib treatment, plasma levels of soluble vascular cell adhesion molecule 1 decreased significantly (P < 0.001), while tissue levels of soluble intercellular adhesion molecule 1 increased (P < 0.01). Some significant correlations between fold changes in certain plasma fibrotic and inflammatory cytokines and changes in clinical parameters after 6 months of treatment were found: patient global scores and IL-13 (r = 0.964, P < 0.0001); ESR and IL-12p70 (r = -0.903, P < 0.01); in tissue samples, patient global score and soluble E-selectin (r = 0.913, P < 0.01); and physician global score with sCD40L (r = -0.883, P < 0.01). CONCLUSION: Some serum and tissue cytokines may have a role in early phase clinical trials of SSc, correlating with changes in clinical parameters. Serum and tissue samples could be analysed in early phase trials to determine whether they support the clinical observations. TRIAL REGISTRATION: http://clinicaltrials.gov/show/NCT01545427.
Assuntos
Benzamidas/uso terapêutico , Citocinas/sangue , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Esclerodermia Difusa/tratamento farmacológico , Humanos , Mesilato de Imatinib , Esclerodermia Difusa/sangue , Esclerodermia Difusa/patologia , Pele/patologia , Resultado do TratamentoRESUMO
Mammalian metallothioneins bind up to seven Zn(2+) ions in two distinct domains: an N-terminal ß-domain that binds three Zn(2+) ions and a C-terminal α-domain that binds four Zn(2+) ions. Domain specificity has been invoked in the metalation mechanism with cluster formation and bridging of the 20 Cys residues taking place prior to saturation with seven Zn(2+) ions. We report a novel experiment that examines Zn(2+) metalation by exploiting the expected decrease in K(F) at the onset of clustering using electrospray ionization mass spectrometry (ESI-MS). During the titration with Zn(2+), the ESI-MS data show that several metalated species coexist until the fully saturated proteins are formed. The relative Zn binding affinities of the seven total sites in the α- and ß-fragments were determined through direct competition for added Zn(2+). The K(F) values for each Zn(2+) are expected to decrease as a function of the remaining available sites and the onset of clustering. Analysis shows that Zn(2+) binds to ß-rhMT with a greater affinity than α-rhMT. The incremental distribution of Zn(2+) between the competing fragments and apo-ßα-rhMT (essentially three and four sites competing with seven sites) identifies the exact point at which clustering begins in the full protein. Analysis of the speciation data shows that Zn(5)-MT forms before clustering begins. This means that all 20 Cys residues of apo-ßα-rhMT are bound terminally to Zn(2+) as [Zn(Cys)(4)](2-) units before clustering begins; there is no domain preference in this first metalation stage. Preferential binding of Zn(2+) to ß- and α-rhMT at the point where ßα-rhMT must form clusters is caused by a significant decrease in the affinity of ßα-rhMT for further Zn(2+). The single-domain Zn(5)-rhMT, in which there are no exposed cysteine sulfurs, is a key component of the metalation pathway because the lower affinities of the two clustered Zn(2+) ions allow donation to apoenzymes.
Assuntos
Metalotioneína/química , Metalotioneína/metabolismo , Zinco/metabolismo , Sequência de Aminoácidos , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Metallothionein is a ubiquitous metal binding protein that plays an important role in metal ion homeostasis and redox chemistry within cells. Mammalian metallothioneins bind a wide variety of metals including the metalloid As3+ in two domains (ß and α) connected by a short linker sequence. Three As3+ bind in each domain for a total of 6 As3+ per protein. In recombinant human metallothionein (rh-MT1a) each As3+ binds three cysteine residues to form As3Cys9(CysSH)2-α-rhMT1a in the 11 Cys α-domain and As3Cys9-ß-rhMT1a in the 9 Cys ß-domain. This means that there should be 2 free cysteines in the α-domain but no free cysteines in the ß-domain. By using benzoquinone, the number and relative accessibility of the free cysteinyl thiols during the metalation reactions were determined. The electrospray ionization mass spectrometry (ESI-MS) data confirmed that each As3+ binds using exactly 3 cysteine thiols and showed that there was a significant difference in the reactivity of the free cysteines during the metalation reaction. After a reaction with two molar equivalents of As3+ to form As2Cys6(CysSH)3-αß-rhMT1a, the remaining 3 Cys in the 9 Cys ß-domain were far less reactive than those in the α-domain. Molecular dynamics calculations for the metalation reactions with As3+ measured by ESI-MS allowed an interpretation of the mass spectral data in terms of the relative location of the cysteine thiols that were not involved in As3+ coordination. Together, these data provide insight into the selection of a specific cysteinyl thiol by the incoming metals during the stepwise metalation of metallothioneins.
Assuntos
Arsênio/química , Cisteína/química , Metalotioneína/química , Sequência de Aminoácidos , Benzoquinonas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Metalotioneína/genética , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometria de Massas por Ionização por Electrospray , Compostos de Sulfidrila/químicaRESUMO
Mammalian metallothioneins (MTs) are a family of small cysteine-rich proteins capable of binding 7 Zn(2+) or Cd(2+) ions into two distinct domains: an N-terminal ß-domain that binds 3 Zn(2+) or Cd(2+) and a C-terminal α-domain that binds 4 Zn(2+) or Cd(2+). MT has been implicated in a number of physiological functions, including metal ion homeostasis, toxic metal detoxification, and as a protective agent against oxidative stress. Conventionally, MT has been understood to coordinate metal ions in a cooperative fashion. Under this mechanism of metalation, the only species of biological relevance would be the metal-free (apo-) form of the protein and the fully metalated (holo-) form of the protein. However, an increasing body of evidence suggests that metalation occurs in a noncooperative manner. If this latter mechanism is correct, then partially metalated forms of the protein will be stable and able to take part in cellular chemistry. We report in this paper conclusive evidence that shows that biologically essential zinc binds to MT in a noncooperative manner. In addition, we report for the first time the stability of a Zn(5)-MT species. The implications of these findings are discussed in terms of the mechanism of metalation.
Assuntos
Apoproteínas/química , Metalotioneína/química , Sulfato de Zinco/metabolismo , Cádmio/metabolismo , Humanos , Modelos MolecularesRESUMO
Mammalian metallothioneins (MTs) are a family of small cysteine rich proteins believed to have a number of physiological functions, including both metal ion homeostasis and toxic metal detoxification. Mammalian MTs bind 7 Zn(2+) or Cd(2+) ions into two distinct domains: an N-terminal ß-domain that binds 3 Zn(2+) or Cd(2+), and a C-terminal α-domain that binds 4 Zn(2+) or Cd(2+). Although stepwise metalation to the saturated M(7)-MT (where M=Zn(2+) or Cd(2+)) species would be expected to take place via a noncooperative mechanism involving the 20 cysteine thiolate ligands, literature reports suggest a cooperative mechanism involving cluster formation prior to saturation of the protein. Electrospray ionization mass spectrometry (ESI-MS) provides this sensitivity through delineation of all species (M(n)-MT, n=0-7) coexisting at each step in the metalation process. We report modeled ESI-mass spectral data for the stepwise metalation of human recombinant MT 1a (rhMT) and its two isolated fractions for three mechanistic conditions: cooperative (where the binding affinities are: K(1)
Assuntos
Cádmio/química , Metalotioneína/química , Modelos Moleculares , Zinco/química , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The metalated forms of metallothionein are well studied (particularly Zn-MT, Cu-MT and Cd-MT), but almost nothing is known about the chemical and structural properties of apometallothioneins despite their importance in initial metalation and subsequent demetalation. Electrospray ionization mass spectrometry was used to provide a detailed view of the structural properties of the metal-free protein. Mass spectra of Zn(7)-MT and apo-MT at pH 7 exhibit the same charge state distribution, indicating that apo-MT is tightly folded like the metallated protein, whereas apo-MT at pH 3 exhibits a charge state spectrum associated with unfolding or denaturation. Benzoquinone was used to modify the cysteines in the ß-MT (9 Bq), and α-MT (11 Bq) fragments, and the full ßα-MT (20 Bq) protein. ESI-MS showed that the overall volume and, therefore, the extent of folding for the modified proteins is similar to that of Zn-MT. Molecular modeling using MM3-MD methods provided the volume of each modified protein. The volumes of the partially modified proteins follow the same trend as the charge states, showing that ESI-MS is an excellent method with which to follow small changes in protein folding as a function of applied chemical stress. The data suggest that the structure of apo-ßα-MT is more organized than previously considered.
Assuntos
Metalotioneína/química , Modelos Moleculares , Benzoquinonas/química , Cisteína/química , Concentração de Íons de Hidrogênio , Metais , Dobramento de Proteína , Estrutura Secundária de Proteína , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Copper(II) coordination by bis(cyclohexanone)oxalyldihydrazone (also known as cuprizone), resulting in the formation of an intensely coloured blue complex, was first reported over 70 years ago. The cuprizone reaction has been employed in colourimetric tests for the presence of trace levels of copper. Cuprizone administration in C57BL/6 mice also leads to demyelination over time - a consequence that appears to be due to copper dyshomeostasis - and this has led to use of cuprizone as the leading method for toxicant-induced generation of an animal model of demyelination since its first use in the 1960s. Despite broad interest in cuprizone and its ability to bind copper there have been relatively few studies to structurally characterize the copper coordination properties of this ligand. In the absence of an aqueous medium, such as neat alcohol, copper and cuprizone exclusively form an amorphous green precipitate. Under aqueous conditions, where a large excess of cuprizone (relative to copper) is present, the blue complex that is synonymous with copper-cuprizone coordination is predominantly formed. The blue and green copper-cuprizone products demonstrate poor solubility and present challenges for conventional structure characterization methods, such as X-ray crystallography or nuclear magnetic resonance spectroscopy. By combining mass spectrometry, X-ray absorption spectroscopy, computational chemistry, and other techniques, a self-consistent picture of the copper coordination structures of the blue and green complexes is revealed - confirming that the blue complex is in the Cu(III) state, containing two hydrolyzed cuprizone ligands per metal centre, while the green complex represents an extended oligomeric complex, comprised of repeating Cu(II) centres that lie 4.8 Å apart and are bridged by unhydrolyzed cuprizone donors.
Assuntos
Complexos de Coordenação , Doenças Desmielinizantes , Animais , Complexos de Coordenação/química , Cobre/química , Cristalografia por Raios X , Cuprizona/efeitos adversos , Doenças Desmielinizantes/induzido quimicamente , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Espectroscopia por Absorção de Raios XRESUMO
The deleterious effects of psychological stress on mainstream T lymphocytes are well documented. However, how stress impacts innate-like T cells is unclear. We report that long-term stress surprisingly abrogates both T helper 1 (TH1)- and TH2-type responses orchestrated by invariant natural killer T (iNKT) cells. This is not due to iNKT cell death because these cells are unusually refractory to stress-inflicted apoptosis. Activated iNKT cells in stressed mice exhibit a "split" inflammatory signature and trigger sudden serum interleukin-10 (IL-10), IL-23, and IL-27 spikes. iNKT cell dysregulation is mediated by cell-autonomous glucocorticoid receptor signaling and corrected upon habituation to predictable stressors. Importantly, under stress, iNKT cells fail to potentiate cytotoxicity against lymphoma or to reduce the burden of metastatic melanoma. Finally, stress physically spares mouse mucosa-associated invariant T (MAIT) cells but hinders their TH1-/TH2-type responses. The above findings are corroborated in human peripheral blood and hepatic iNKT/MAIT cell cultures. Our work uncovers a mechanism of stress-induced immunosuppression.
Assuntos
Neoplasias Hepáticas/imunologia , Linfoma/imunologia , Células T Invariantes Associadas à Mucosa/imunologia , Células T Matadoras Naturais/imunologia , Estresse Psicológico/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Linhagem Celular Tumoral , Doença Crônica , Corticosterona/farmacologia , Citotoxicidade Imunológica , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imobilização , Imunidade Inata , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-23/genética , Interleucina-23/imunologia , Interleucinas/genética , Interleucinas/imunologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Linfoma/genética , Linfoma/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Células T Invariantes Associadas à Mucosa/efeitos dos fármacos , Células T Invariantes Associadas à Mucosa/patologia , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/patologia , Metástase Neoplásica , Oxidopamina/farmacologia , Transdução de Sinais , Estresse Psicológico/genética , Estresse Psicológico/patologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/patologia , Equilíbrio Th1-Th2RESUMO
8-Hydroxyquinolines (8HQs) comprise a family of metal-binding compounds that have been used or tested for use in numerous medicinal applications, including as treatments for bacterial infection, Alzheimer's disease, and cancer. Two key 8HQs, CQ (5-chloro-7-iodo-8-hydroxyquinoline) and PBT2 (2-(dimethylamino)methyl-5,7-dichloro-8-hydroxyquinoline), have drawn considerable interest and have been the focus of many studies investigating their in vivo properties. These drugs have been described as copper and zinc ionophores because they do not cause metal depletion, as would be expected for a chelation mechanism, but rather cellular accumulation of these ions. In studies of their anti-cancer properties, CQ has been proposed to elicit toxic intracellular copper accumulation and to trigger apoptotic cancer cell death through several possible pathways. In this study we used synchrotron X-ray fluorescence imaging, in combination with biochemical assays and light microscopy, to investigate 8HQ-induced alterations to metal ion homeostasis, as well as cytotoxicity and cell death. We used the bromine fluorescence from a bromine labelled CQ congener (5,7-dibromo-8-hydroxyquinoline; B2Q) to trace the intracellular localization of B2Q following treatment and found that B2Q crosses the cell membrane. We also found that 8HQ co-treatment with Cu(ii) results in significantly increased intracellular copper and significant cytotoxicity compared with 8HQ treatments alone. PBT2 was found to be more cytotoxic, but a weaker Cu(ii) ionophore than other 8HQs. Moreover, treatment of cells with copper in the presence of CQ or B2Q resulted in copper accumulation in the nuclei, while PBT2-guided copper was distributed near to the cell membrane. These results suggest that PBT2 may be acting through a different mechanism than that of other 8HQs to cause the observed cytotoxicity.