RESUMO
Human serum retinol-binding protein (RBP) is a plasma transport protein for vitamin A. RBP is a prime subclass of lipocalins, which bind nonpolar ligands within a ß-barrel. To understand the role of Trp 24, one of the highly conserved residues in RBP, free energy simulations have been carried out to understand the effects of the mutations from Trp at position 24 to Leu, Phe, and Tyr in the apo-RBP on its thermal stability. We examine various unfolded systems to study the dependence of the free energy differences on the denatured structure. Our calculated free energy difference values for the three mutations are in excellent agreement with the experimental values when the initial coordinates of the seven-residue peptide segments truncated from the crystal structure are used for the denatured systems. Our free energy change differences for the TrpâLeu, TrpâPhe, and TrpâTyr mutations are 2.50 ± 0.69, 2.58 ± 0.50, and 2.49 ± 0.48 kcal/mol, respectively, when the native-like seven-residue peptides are used as models for the denatured systems. The main contributions to the free energy change differences for the Trp24âLeu and Trp24âPhe mutations are mainly from van der Waals and covalent interactions, respectively. Electrostatic, van der Waals and covalent terms equally contribute to the free energy change difference for the Trp24âTyr mutation. The free energy simulation helps understand the detailed microscopic mechanism of the stability of the RBP mutants relative to the wild type and the role of the highly conserved residue, Trp24, of the human RBP.Communicated by Ramaswamy H. Sarma.
Assuntos
Proteínas de Transporte , Vitamina A , Humanos , Proteínas de Transporte/química , Mutação , Peptídeos/metabolismo , Proteínas de Ligação ao Retinol/química , Proteínas de Ligação ao Retinol/genética , Proteínas de Ligação ao Retinol/metabolismoRESUMO
Interphotoreceptor retinoid-binding protein (IRBP) is a highly expressed protein secreted by rod and cone photoreceptors that has major roles in photoreceptor homeostasis as well as retinoid and polyunsaturated fatty acid transport between the neural retina and retinal pigment epithelium. Despite two crystal structures reported on fragments of IRBP and decades of research, the overall structure of IRBP and function within the visual cycle remain unsolved. Here, we studied the structure of native bovine IRBP in complex with a monoclonal antibody (mAb5) by cryo-electron microscopy, revealing the tertiary and quaternary structure at sufficient resolution to clearly identify the complex components. Complementary mass spectrometry experiments revealed the structure and locations of N-linked carbohydrate post-translational modifications. This work provides insight into the structure of IRBP, displaying an elongated, flexible three-dimensional architecture not seen among other retinoid-binding proteins. This work is the first step in elucidation of the function of this enigmatic protein.
Assuntos
Proteínas do Olho/química , Proteínas de Ligação ao Retinol/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/química , Bovinos , Microscopia Crioeletrônica , Proteínas do Olho/imunologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação ao Retinol/imunologia , Imagem Individual de MoléculaRESUMO
Quantum chemical calculations are important for elucidating light-capturing mechanisms in photobiological systems. The time-dependent density functional theory (TDDFT) has become a popular methodology because of its balance between accuracy and computational scaling, despite its problems in describing, for example, charge transfer states. As a step toward systematically understanding the performance of TDDFT calculations on biomolecular systems, we study here 17 commonly used density functionals, including seven long-range separated functionals, and compare the obtained results with excitation energies calculated at the approximate second order coupled-cluster theory level (CC2). The benchmarking set includes the first five singlet excited states of 11 chemical analogues of biochromophores from the green fluorescent protein, rhodopsin/bacteriorhodopsin (Rh/bR), and the photoactive yellow protein. We find that commonly used pure density functionals such as BP86, PBE, M11-L, and hybrid functionals with 20-25% of Hartree-Fock (HF) exchange (B3LYP, PBE0) have a tendency to consistently underestimate vertical excitation energies (VEEs) relative to the CC2 values, whereas hybrid density functionals with around 50% HF exchange such as BHLYP, PBE50, and M06-2X and long-range corrected functionals such as CAM-B3LYP, ωPBE, ωPBEh, ωB97X, ωB97XD, BNL, and M11 overestimate the VEEs. We observe that calculations using the CAM-B3LYP and ωPBEh functionals with 65% and 100% long-range HF exchange, respectively, lead to an overestimation of the VEEs by 0.2-0.3 eV for the benchmarking set. To reduce the systematic error, we introduce here two new empirical functionals, CAMh-B3LYP and ωhPBE0, for which we adjusted the long-range HF exchange to 50%. The introduced parameterization reduces the mean signed average (MSA) deviation to 0.07 eV and the root mean square (rms) deviation to 0.17 eV as compared to the CC2 values. In the present study, TDDFT calculations using the aug-def2-TZVP basis sets, the best performing functionals relative to CC2 are ωhPBE0 (rms = 0.17, MSA = 0.06 eV); CAMh-B3LYP (rms = 0.16, MSA = 0.07 eV); and PBE0 (rms = 0.23, MSA = -0.14 eV). For the popular range-separated CAM-B3LYP functional, we obtain an rms value of 0.31 eV and an MSA value of 0.25 eV, which can be compared with the rms and MSA values of 0.37 and -0.31 eV, respectively, as obtained at the B3LYP level.
Assuntos
Teoria da Densidade Funcional , Proteínas de Fluorescência Verde/química , Proteínas de Ligação ao Retinol/química , Algoritmos , Bases de Dados de Proteínas , Humanos , Modelos MolecularesRESUMO
Retinol binding protein IV (RBP) functions as the principal carrier of retinol (Vitamin A) in the blood, where RBP circulates bound to another serum protein, transthyretin. Isolation of pure RBP from the transthyretin complex in human serum can be difficult, but expression of RBP in recombinant systems can circumvent these purification issues. Human recombinant RBP has previously been successfully expressed and purified from E. coli, but recovery of active protein typically requires extensive processing steps, such as denaturing and refolding, and complex purification steps, such as multi-modal chromatography. Furthermore, these methods produce recombinant proteins, often tagged, that display different functional and structural characteristics across systems. In this work, we optimized downstream processing by use of an intein-based expression system in E. coli to produce tag-free, human recombinant RBP (rRBP) with intact native amino termini at yields of up to ~15 mg/L off column. The novel method requires solubilization of inclusion bodies and subsequent oxidative refolding in the presence of retinol, but importantly allows for one-step chromatographic purification that yields high purity rRBP with no N-terminal Met or other tag. Previously reported purification methods typically require two or more chromatographic separation steps to recover tag-free rRBP. Given the interest in mechanistic understanding of RBP transport of retinol in health and disease, we characterized our purified product extensively to confirm rRBP is both structurally and functionally a suitable replacement for serum-derived RBP.
Assuntos
Proteínas Recombinantes/isolamento & purificação , Proteínas de Ligação ao Retinol/biossíntese , Proteínas de Ligação ao Retinol/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Humanos , Inteínas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas de Ligação ao Retinol/química , Vitamina A/sangue , Vitamina A/químicaRESUMO
The chrysanthemum foliar nematode (CFN), Aphelenchoides ritzemabosi, is a migratory, plant-parasitic nematode that is widely distributed and infects the aboveground parts of many plants. The fatty acid- and retinoid-binding proteins (FAR) are nematode-specific proteins that are involved in the development, reproduction, and infection of nematodes and are secreted into the tissues to disrupt the plant defense reaction. In this study, we obtained the full-length sequence of the FAR gene (Ar-far-1) from CFN, which is 727 bp and includes a 546 bp ORF that encodes 181 amino acids. Ar-FAR-1 from CFN has the highest sequence similarity to Ab-FAR-1 from A. besseyi, and they are located within the same branch of the phylogenetic tree. Fluorescence-based ligand-binding analysis confirmed that recombinant Ar-FAR-1 was bound to fatty acids and retinol. Ar-far-1 mRNA was expressed in the muscle layer, intestine, female genital system, and egg of CFN, and more highly expressed in females than in males among the four developmental stages of CFN. We demonstrated that the reproduction number and infection capacity of CFN decreased significantly when Ar-far-1 was effectively silenced by in vitro RNAi. Ar-far-1 plays an important role in the development, reproduction, infectivity, and pathogenesis of CFN and may be used as an effective target gene for the control of CFN. The results provide meaningful data about the parasitic and pathogenic genes of CFN to study the interaction mechanism between plant-parasitic nematodes and hosts.
Assuntos
Proteínas de Ligação a Ácido Graxo , Genes de Helmintos , Proteínas de Helminto , Proteínas de Ligação ao Retinol , Rabditídios , Animais , Chrysanthemum/parasitologia , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Doenças das Plantas/parasitologia , Folhas de Planta/parasitologia , Proteínas de Ligação ao Retinol/química , Proteínas de Ligação ao Retinol/genética , Proteínas de Ligação ao Retinol/metabolismo , Rabditídios/química , Rabditídios/genética , Rabditídios/metabolismoRESUMO
Lipid-binding proteins (LBPs) are soluble proteins responsible for the uptake, transport, and storage of a large variety of hydrophobic lipophilic molecules including fatty acids, steroids, and other lipids in the cellular environment. Among the LBPs, fatty acid binding proteins (FABPs) present preferential binding affinities for long-chain fatty acids. While most of FABPs in vertebrates and invertebrates present similar ß-barrel structures with ligands accommodated in their central cavity, parasitic nematode worms exhibit additional unusual α-helix rich fatty acid- and retinol-binding proteins (FAR). Herein, we report the comparison of extended molecular dynamics (MD) simulations performed on the ligand-free and palmitic acid-bond states of the Necator americanus FAR-1 (Na-FAR-1) with respect to other classical ß-barrel FABPs. Principal component analysis (PCA) has been used to identify the different conformations adopted by each system during MD simulations. The α-helix fold encompasses a complex internal ligand-binding cavity with a remarkable conformational plasticity that allows reversible switching between distinct states in the holo-Na-FAR-1. The cavity can change up to one-third of its size affected by conformational changes of the protein-ligand complex. Besides, the ligand inside the cavity is not fixed but experiences large conformational changes between bent and stretched conformations. These changes in the ligand conformation follow changes in the cavity size dictated by the transient protein conformation. On the contrary, protein-ligand complex in ß-barrel FABPs fluctuates around a unique conformation. The significantly more flexible holo-Na-FAR-1 ligand-cavity explains its larger ligand multiplicity respect to ß-barrel FABPs.
Assuntos
Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/metabolismo , Simulação de Dinâmica Molecular , Proteínas de Ligação ao Retinol/química , Proteínas de Ligação ao Retinol/metabolismo , Ligantes , Conformação ProteicaRESUMO
The Joslin Medalist Study characterized people affected with type 1 diabetes for 50 years or longer. More than 35% of these individuals exhibit no to mild diabetic retinopathy (DR), independent of glycemic control, suggesting the presence of endogenous protective factors against DR in a subpopulation of patients. Proteomic analysis of retina and vitreous identified retinol binding protein 3 (RBP3), a retinol transport protein secreted mainly by the photoreceptors, as elevated in Medalist patients protected from advanced DR. Mass spectrometry and protein expression analysis identified an inverse association between vitreous RBP3 concentration and DR severity. Intravitreal injection and photoreceptor-specific overexpression of RBP3 in rodents inhibited the detrimental effects of vascular endothelial growth factor (VEGF). Mechanistically, our results showed that recombinant RBP3 exerted the therapeutic effects by binding and inhibiting VEGF receptor tyrosine phosphorylation. In addition, by binding to glucose transporter 1 (GLUT1) and decreasing glucose uptake, RBP3 blocked the detrimental effects of hyperglycemia in inducing inflammatory cytokines in retinal endothelial and Müller cells. Elevated expression of photoreceptor-secreted RBP3 may have a role in protection against the progression of DR due to hyperglycemia by inhibiting glucose uptake via GLUT1 and decreasing the expression of inflammatory cytokines and VEGF.
Assuntos
Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Proteínas do Olho/metabolismo , Retina/metabolismo , Retina/patologia , Proteínas de Ligação ao Retinol/metabolismo , 3-O-Metilglucose/metabolismo , Ácidos/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Desoxiglucose/metabolismo , Diabetes Mellitus/fisiopatologia , Retinopatia Diabética/fisiopatologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Ependimogliais/efeitos dos fármacos , Células Ependimogliais/metabolismo , Proteínas do Olho/administração & dosagem , Proteínas do Olho/sangue , Proteínas do Olho/química , Glicólise/efeitos dos fármacos , Humanos , Injeções Intravítreas , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Substâncias Protetoras/farmacologia , Domínios Proteicos , Ratos Endogâmicos Lew , Proteínas Recombinantes/farmacologia , Reprodutibilidade dos Testes , Retina/fisiopatologia , Proteínas de Ligação ao Retinol/administração & dosagem , Proteínas de Ligação ao Retinol/química , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Corpo Vítreo/efeitos dos fármacos , Corpo Vítreo/metabolismoRESUMO
Parasitic nematodes produce an unusual class of fatty acid and retinol (FAR)-binding proteins that may scavenge host fatty acids and retinoids. Two FARs from Brugia malayi (Bm-FAR-1 and Bm-FAR-2) were expressed as recombinant proteins, and their ligand binding, structural characteristics, and immunogenicities examined. Circular dichroism showed that rBm-FAR-1 and rBm-FAR-2 are similarly rich in α-helix structure. Unexpectedly, however, their lipid binding activities were found to be readily differentiated. Both FARs bound retinol and cis-parinaric acid similarly, but, while rBm-FAR-1 induced a dramatic increase in fluorescence emission and blue shift in peak emission by the fluorophore-tagged fatty acid (dansyl-undecanoic acid), rBm-FAR-2 did not. Recombinant forms of the related proteins from Onchocerca volvulus, rOv-FAR-1 and rOv-FAR-2, were found to be similarly distinguishable. This is the first FAR-2 protein from parasitic nematodes that is being characterized. The relative protein abundance of Bm-FAR-1 was higher than Bm-FAR-2 in the lysates of different developmental stages of B. malayi. Both FAR proteins were targets of strong IgG1, IgG3 and IgE antibody in infected individuals and individuals who were classified as endemic normal or putatively immune. In a B. malayi infection model in gerbils, immunization with rBm-FAR-1 and rBm-FAR-2 formulated in a water-in-oil-emulsion (®Montanide-720) or alum elicited high titers of antigen-specific IgG, but only gerbils immunized with rBm-FAR-1 formulated with the former produced a statistically significant reduction in adult worms (68%) following challenge with B. malayi infective larvae. These results suggest that FAR proteins may play important roles in the survival of filarial nematodes in the host, and represent potential candidates for vaccine development against lymphatic filariasis and related filarial infections.
Assuntos
Antígenos de Helmintos/imunologia , Brugia Malayi/imunologia , Proteínas de Ligação a Ácido Graxo/imunologia , Filariose/prevenção & controle , Proteínas de Ligação ao Retinol/imunologia , Vacinas Sintéticas/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/química , Dicroísmo Circular , Modelos Animais de Doenças , Proteínas de Ligação a Ácido Graxo/química , Feminino , Gerbillinae , Humanos , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Masculino , Carga Parasitária , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas de Ligação ao Retinol/química , Resultado do Tratamento , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/isolamento & purificação , Vitamina A/metabolismoRESUMO
ß-Amyloid (Aß) aggregation is causally linked to Alzheimer's disease. On the basis of in vitro and transgenic animal studies, transthyretin (TTR) is hypothesized to provide neuroprotection against Aß toxicity by binding to Aß and inhibiting its aggregation. TTR is a homotetrameric protein that circulates in blood and cerebrospinal fluid; its normal physiological role is as a carrier for thyroxine and retinol-binding protein (RBP). RBP forms a complex with retinol, and the holoprotein (hRBP) binds with high affinity to TTR. In this study, the role of TTR ligands in TTR-mediated inhibition of Aß aggregation was investigated. hRBP strongly reduced the ability of TTR to inhibit Aß aggregation. The effect was not due to competition between Aß and hRBP for binding to TTR, as Aß bound equally well to TTR-hRBP complexes and TTR. hRBP is known to stabilize the TTR tetrameric structure. We show that Aß partially destabilizes TTR and that hRBP counteracts this destabilization. Taken together, our results support a mechanism wherein TTR-mediated inhibition of Aß aggregation requires not only TTR-Aß binding but also destabilization of TTR quaternary structure.
Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Pré-Albumina/química , Proteínas de Ligação ao Retinol/química , Humanos , Ligantes , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de ProteínaRESUMO
Retinoids are a class of compounds derived from vitamin A or having structural and/or functional similarities with vitamin A. They are classified into three generations based on their molecular structures. Inside the body, retinoids bind to several classes of proteins including retinoid-binding proteins and retinoid nuclear receptors. This eventually leads to the activation of specific regulatory regions of DNA - called the retinoic acid response elements - involved in regulating cell growth, differentiation and apoptosis. Several clinical trials have studied the role of topical and systemic retinoids in disease, and research is still ongoing. Currently, retinoids are used in several fields of medicine. This paper aims to review the structure, mechanisms of action, and adverse effects of retinoids, as well as some of their current uses in Dermatology.
Assuntos
Retinoides/metabolismo , Dermatopatias/tratamento farmacológico , Acne Vulgar/tratamento farmacológico , Acne Vulgar/patologia , Administração Tópica , Infecções Bacterianas/etiologia , Humanos , Psoríase/tratamento farmacológico , Psoríase/patologia , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides/química , Receptores X de Retinoides/metabolismo , Retinoides/efeitos adversos , Retinoides/uso terapêutico , Proteínas de Ligação ao Retinol/química , Proteínas de Ligação ao Retinol/metabolismo , Dermatopatias/patologia , Vitamina A/efeitos adversos , Vitamina A/química , Vitamina A/metabolismo , Xeroftalmia/etiologiaRESUMO
Vitamin A homeostasis is critical to normal cellular function. Retinol-binding protein (RBP) is the sole specific carrier in the bloodstream for hydrophobic retinol, the main form in which vitamin A is transported. The integral membrane receptor STRA6 mediates cellular uptake of vitamin A by recognizing RBP-retinol to trigger release and internalization of retinol. We present the structure of zebrafish STRA6 determined to 3.9-angstrom resolution by single-particle cryo-electron microscopy. STRA6 has one intramembrane and nine transmembrane helices in an intricate dimeric assembly. Unexpectedly, calmodulin is bound tightly to STRA6 in a noncanonical arrangement. Residues involved with RBP binding map to an archlike structure that covers a deep lipophilic cleft. This cleft is open to the membrane, suggesting a possible mode for internalization of retinol through direct diffusion into the lipid bilayer.
Assuntos
Proteínas de Membrana/química , Proteínas de Membrana Transportadoras/química , Proteínas de Ligação ao Retinol/química , Vitamina A/metabolismo , Proteínas de Peixe-Zebra/química , Animais , Transporte Biológico , Cálcio/química , Calmodulina/química , Microscopia Crioeletrônica , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas de Ligação ao Retinol/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
A gene encoding fatty acid- and retinoid-binding protein was isolated from the cereal cyst nematode Heterodera avenae and the biochemical function of the protein that it encodes was analysed. The full-length cDNA of the Ha-far-1 gene is 827 bp long and includes a 22- nucleotide trans-spliced leader sequence (SL1) at its 5-end. The genomic clone of Ha-far-1 consists of eight exons separated by seven introns, which range in size from 48 to 186 bp. The Ha-far-1 cDNA contains an open reading frame encoding a 191 amino acid protein, with a predicted secretory signal peptide. Sequence analysis showed that Ha-FAR-1 has highest similarity to the Gp-FAR-1 protein from the potato cyst nematode, Globodera pallida and that the protein was grouped with all homologues from other plant-parasitic nematodes in a phylogenetic analysis. Fluorescence-based ligand binding analysis confirmed that the recombinant Ha-FAR-1 protein was able to bind fatty acids and retinol. Spatial and temporal expression assays showed that the transcripts of Ha-far-1 accumulated mainly in the hypodermis and that the gene is most highly expressed in third-stage juveniles of H. avenae. Fluorescence immunolocalization showed that the Ha-FAR-1 protein was present on the surface of the infective second-stage juveniles of H. avenae. Nematodes treated with dsRNA corresponding to Ha-far-1 showed significantly reduced reproduction compared to nematodes exposed to dsRNA from a non-endogenous gene, suggesting that Ha-far-1 may be an effective target gene for control of H. avenae using an RNAi strategy.
Assuntos
Proteínas de Ligação a Ácido Graxo/isolamento & purificação , Proteínas de Helminto/isolamento & purificação , Proteínas de Ligação ao Retinol/isolamento & purificação , Tylenchoidea/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Clonagem Molecular , DNA Complementar/química , DNA de Helmintos/isolamento & purificação , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Proteínas de Helminto/química , Proteínas de Helminto/genética , Hibridização In Situ , Ligantes , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Ligação ao Retinol/química , Proteínas de Ligação ao Retinol/genética , Alinhamento de Sequência , Transcrição Gênica , Triticum/parasitologia , Tylenchoidea/genéticaRESUMO
Multiple functions for Interphotoreceptor Retinoid-Binding Protein (IRBP) may explain its localization in the retina, vitreous and pineal gland and association with retinitis pigmentosa and myopia. We have been engaged in uncovering the structure-function relationships of this interesting protein long thought to bind visual-cycle retinoids and fatty acids in the subretinal space. Although hydrophobic domains capable of binding such ligands have now been found, we ask what other structural domains might be present that could predict new functions? Interestingly, IRBP possesses a fold similar to C-terminal processing proteases (CTPases) but is missing the PDZ domain. Here we present structural evidence that this fold may have a role in a recently observed autoproteolytic activity of the two-module zebrafish (z) IRBP (Ghosh et al. Exp. Eye Res., 2015). When the structure of Scenedesmus obliquus D1 CTPase (D1P) is superimposed with the first module of zIRBP (z1), the PDZ domain of D1P occupies roughly the same position in the amino acid sequence as the inter-domain tether in z1, between residues P71 and P85. The catalytic triad K397, S372 and E375 of D1P is located at the inter-domain interfacial cleft, similarly as the tetrad K241, S243, D177 and T179 of z1 residues, presumed to have proteolytic function. Packing of two adjacent symmetry-related molecules within the z1 crystal show that the helix α8 penetrates the interfacial cleft underneath the inter-domain tether, forming a simple intermolecular "knot". The full-length zIRBP is cleaved at or immediately after T309, which is located at the end of α8 and is the ninth residue of the second module z2. We propose that the helix α8 within intact zIRBP bends at P301, away from the improbable knotted fold, and positions the cleavage site T309 near the putative catalytic tetrad of the neighboring zIRBP to be proteolytically cleaved. The conservation of this functional catalytic domain suggests that possible physiological roles of IRBP as a hydrolase needs to be considered.
Assuntos
Proteínas do Olho/química , Dobramento de Proteína , Proteínas de Ligação ao Retinol/química , Proteínas de Peixe-Zebra/química , Sequência de Aminoácidos , Animais , Endopeptidases/química , Proteínas do Olho/fisiologia , Ligantes , Proteólise , Retina/metabolismo , Proteínas de Ligação ao Retinol/fisiologia , Espectrometria de Fluorescência , Difração de Raios X , Peixe-Zebra , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Interphotoreceptor retinoid-binding protein (IRBP) has a remarkable role in targeting and protecting all-trans and 11-cis retinol, and 11-cis retinal during the rod and cone visual cycles. Little is known about how the correct retinoid is efficiently delivered and removed from the correct cell at the required time. It has been proposed that different fatty composition at that the outer-segments and retinal-pigmented epithelium have an important role is regulating the delivery and uptake of the visual cycle retinoids at the cell-interphotoreceptor-matrix interface. Although this suggests intriguing mechanisms for the role of local fatty acids in visual-cycle retinoid trafficking, nothing is known about the structural basis of IRBP-fatty acid interactions. Such regulation may be mediated through IRBP's unusual repeating homologous modules, each containing about 300 amino acids. We have been investigating structure-function relationships of Zebrafish IRBP (zIRBP), which has only two tandem modules (z1 and z2), as a model for the more complex four-module mammalian IRBP's. Here we report the first X-ray crystal structure of a teleost IRBP, and the only structure with a bound ligand. The X-ray structure of z1, determined at 1.90 Å resolution, reveals a two-domain organization of the module (domains A and B). A deep hydrophobic pocket with a single bound molecule of oleic acid was identified within the N-terminal domain A. In fluorescence titrations assays, oleic acid displaced all-trans retinol from zIRBP. Our study, which provides the first structure of an IRBP with bound ligand, supports a potential role for fatty acids in regulating retinoid binding.
Assuntos
Proteínas do Olho/química , Conformação Proteica , Proteínas de Ligação ao Retinol/química , Proteínas de Peixe-Zebra/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Proteínas do Olho/metabolismo , Ligantes , Dados de Sequência Molecular , Ácido Oleico/química , Ácido Oleico/metabolismo , Proteínas de Ligação ao Retinol/metabolismo , Espectrometria de Fluorescência , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Fatty acid and retinol-binding proteins (FARs) comprise a family of unusual α-helix rich lipid-binding proteins found exclusively in nematodes. They are secreted into host tissues by parasites of plants, animals and humans. The structure of a FAR protein from the free-living nematode Caenorhabditis elegans is available, but this protein [C. elegans FAR-7 (Ce-FAR-7)] is from a subfamily of FARs that does not appear to be important at the host/parasite interface. We have therefore examined [Necator americanus FAR-1 (Na-FAR-1)] from the blood-feeding intestinal parasite of humans, N. americanus. The 3D structure of Na-FAR-1 in its ligand-free and ligand-bound forms, determined by NMR (nuclear magnetic resonance) spectroscopy and X-ray crystallography respectively, reveals an α-helical fold similar to Ce-FAR-7, but Na-FAR-1 possesses a larger and more complex internal ligand-binding cavity and an additional C-terminal α-helix. Titration of apo-Na-FAR-1 with oleic acid, analysed by NMR chemical shift perturbation, reveals that at least four distinct protein-ligand complexes can be formed. Na-FAR-1 and possibly other FARs may have a wider repertoire for hydrophobic ligand binding, as confirmed in the present study by our finding that a range of neutral and polar lipids co-purify with the bacterially expressed recombinant protein. Finally, we show by immunohistochemistry that Na-FAR-1 is present in adult worms with a tissue distribution indicative of possible roles in nutrient acquisition by the parasite and in reproduction in the male.
Assuntos
Interações Hospedeiro-Parasita , Necator americanus/metabolismo , Necatoríase/metabolismo , Proteínas de Ligação ao Retinol/metabolismo , Animais , Sítios de Ligação , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/patogenicidade , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Ligantes , Necator americanus/química , Necator americanus/patogenicidade , Necatoríase/parasitologia , Reprodução , Proteínas de Ligação ao Retinol/químicaRESUMO
We aimed to study the role of the nucleotide receptor P2Y2R in the development of experimental autoimmune uveitis (EAU). EAU was induced in P2Y2+/+ and P2Y2-/- mice by immunization with IRBP peptide or by adoptive transfer of in vitro restimulated semi-purified IRBP-specific enriched T lymphocytes from spleens and lymph nodes isolated from native C57Bl/6 or P2Y2+/+ and P2Y2-/- immunized mice. Clinical and histological scores were used to grade disease severity. Splenocytes and lymph node cell phenotypes were analyzed using flow cytometry. Semi-purified lymphocytes and MACS-purified CD4+ T lymphocytes from P2Y2+/+ and P2Y2-/- immunized mice were tested for proliferation and cytokine secretion. Our data show that clinical and histological scores were significantly decreased in IRBP-immunized P2Y2-/- mice as in P2Y2-/- mice adoptively transfered with enriched T lymphocytes from C57Bl/6 IRBP-immunized mice. In parallel, naïve C57Bl/6 mice adoptively transferred with T lymphocytes from P2Y2-/- IRBP-immunized mice also showed significantly less disease. No differences in term of spleen and lymph node cell recruitment or phenotype appeared between P2Y2-/- and P2Y2+/+ immunized mice. However, once restimulated in vitro with IRBP, P2Y2-/- T cells proliferate less and secrete less cytokines than the P2Y2+/+ one. We further found that antigen-presenting cells of P2Y2-/- immunized mice were responsible for this proliferation defect. Together our data show that P2Y2-/- mice are less susceptible to mount an autoimmune response against IRBP. Those results are in accordance with the danger model, which makes a link between autoreactive lymphocyte activation, cell migration and the release of danger signals such as extracellular nucleotides.
Assuntos
Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Receptores Purinérgicos P2Y2/deficiência , Uveíte/imunologia , Uveíte/metabolismo , Transferência Adotiva , Sequência de Aminoácidos , Animais , Células Apresentadoras de Antígenos/imunologia , Proliferação de Células , Citocinas/metabolismo , Proteínas do Olho/química , Técnicas de Inativação de Genes , Humanos , Imunização , Linfonodos/imunologia , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Receptores Purinérgicos P2Y2/genética , Proteínas de Ligação ao Retinol/química , Baço/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Strategies to modify nanoparticles with biological ligands for targeted drug delivery in vivo have been widely studied but met with limited clinical success. A possible reason is that, in the blood circulation, serum proteins could rapidly form a layer of protein "corona" on the vehicle surface, which might block the modified ligands and hamper their targeting functions. We speculate that strategies for drug delivery can be designed based upon elegant control of the corona formation on the vehicle surfaces. In this study, we demonstrate a retinol-conjugated polyetherimine (RcP) nanoparticle system that selectively recruited the retinol binding protein 4 (RBP) in its corona components. RBP was found to bind retinol, and direct the antisense oligonucleotide (ASO)-laden RcP carrier to hepatic stellate cells (HSC), which play essential roles in the progression of hepatic fibrosis. In both mouse fibrosis models, induced by carbon tetrachloride (CCl4) and bile duct ligation (BDL), respectively, the ASO-laden RcP particles effectively suppressed the expression of type I collagen (collagen I), and consequently ameliorated hepatic fibrosis. Such findings suggest that this delivery system, designed to exploit the power of corona proteins, can serve as a promising tool for targeted delivery of therapeutic agents for the treatment of hepatic fibrosis.
Assuntos
Portadores de Fármacos/química , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/terapia , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/genética , Coroa de Proteína/química , Albuminas/química , Animais , Linhagem Celular , Humanos , Cirrose Hepática/genética , Masculino , Camundongos , Nanopartículas/química , Oligonucleotídeos Antissenso/farmacocinética , Polietilenoimina/química , Proteínas de Ligação ao Retinol/química , Distribuição Tecidual , Transfecção , Vitamina A/químicaRESUMO
Retinol degrades rapidly in light into a variety of photoproducts. It is remarkable that visual cycle retinoids can evade photodegradation as they are exchanged between the photoreceptors, retinal pigment epithelium and Müller glia. Within the interphotoreceptor matrix, all-trans retinol, 11-cis retinol and retinal are bound by interphotoreceptor retinoid-binding protein (IRBP). Apart from its role in retinoid trafficking and targeting, could IRBP have a photoprotective function? HPLC was used to evaluate the ability of IRBP to protect all-trans and 11-cis retinols from photodegradation when exposed to incandescent light (0 to 8842 µW cm(-2)); time periods of 0-60 min, and bIRBP: retinol molar ratios of 1:1 to 1:5. bIRBP afforded a significant prevention of both all-trans and 11-cis retinol to rapid photodegradation. The effect was significant over the entire light intensity range tested, and extended to the bIRBP: retinol ratio 1:5. In view of the continual exposure of the retina to light, and the high oxidative stress in the outer retina, our results suggest IRBP may have an important protective role in the visual cycle by reducing photodegradation of all-trans and 11-cis retinols. This role of IRBP is particularly relevant in the high flux conditions of the cone visual cycle.
Assuntos
Proteínas do Olho/química , Protetores contra Radiação/química , Retinaldeído/química , Proteínas de Ligação ao Retinol/química , Vitamina A/química , Animais , Bovinos , Relação Dose-Resposta à Radiação , Proteínas do Olho/isolamento & purificação , Luz , Fotólise , Protetores contra Radiação/isolamento & purificação , Retina/química , Retina/efeitos da radiação , Proteínas de Ligação ao Retinol/isolamento & purificaçãoRESUMO
Vitamin A plays pleiotropic roles in the immune system. A recent eLife paper by Hooper and colleagues shows that hepatic and intestinal serum amyloid A proteins, which are induced in response to infection, can transport vitamin A metabolites to tissues and thus impact immune responses both locally and systemically.
Assuntos
Proteínas de Ligação ao Retinol/química , Infecções por Salmonella/metabolismo , Proteína Amiloide A Sérica/química , Vitamina A/metabolismo , Animais , HumanosRESUMO
During inflammation, serum amyloid A proteins transport retinol to infected tissues.