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1.
Cells ; 8(6)2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31226822

RESUMO

Worldwide, there is a rise in the prevalence of allergic diseases, and novel efficient therapeutic approaches are still needed to alleviate disease burden. Prostaglandin D2 (PGD2) has emerged as a central inflammatory lipid mediator associated with increased migration, activation and survival of leukocytes in various allergy-associated disorders. In the periphery, the hematopoietic PGD synthase (hPGDS) acts downstream of the arachidonic acid/COX pathway catalysing the isomerisation of PGH2 to PGD2, which makes it an interesting target to treat allergic inflammation. Although much effort has been put into developing efficient hPGDS inhibitors, no compound has made it to the market yet, which indicates that more light needs to be shed on potential PGD2 sources and targets to determine which particular condition and patient will benefit most and thereby improve therapeutic efficacy. In this review, we want to revisit current knowledge about hPGDS function, expression in allergy-associated cell types and their contribution to PGD2 levels as well as beneficial effects of hPGDS inhibition in allergic asthma, rhinitis, atopic dermatitis, food allergy, gastrointestinal allergic disorders and anaphylaxis.


Assuntos
Hematopoese , Hipersensibilidade/tratamento farmacológico , Inflamação/tratamento farmacológico , Oxirredutases Intramoleculares/uso terapêutico , Lipocalinas/uso terapêutico , Animais , Ensaios Clínicos como Assunto , Humanos , Hipersensibilidade/complicações , Inflamação/complicações , Oxirredutases Intramoleculares/química , Lipocalinas/química , Prostaglandina D2/química , Prostaglandina D2/metabolismo
2.
Bioorg Med Chem ; 27(8): 1456-1478, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30858025

RESUMO

With the goal of discovering more selective anti-inflammatory drugs, than COX inhibitors, to attenuate prostaglandin signaling, a fragment-based screen of hematopoietic prostaglandin D synthase was performed. The 76 crystallographic hits were sorted into similar groups, with the 3-cyano-quinoline 1a (FP IC50 = 220,000 nM, LE = 0.43) being a potent member of the 6,6-fused heterocyclic cluster. Employing SAR insights gained from structural comparisons of other H-PGDS fragment binding mode clusters, the initial hit 1a was converted into the 70-fold more potent quinoline 1d (IC50 = 3,100 nM, LE = 0.49). A systematic substitution of the amine moiety of 1d, utilizing structural information and array chemistry, with modifications to improve inhibitor stability, resulted in the identification of the 300-fold more active H-PGDS inhibitor tool compound 1bv (IC50 = 9.9 nM, LE = 0.42). This selective inhibitor exhibited good murine pharmacokinetics, dose-dependently attenuated PGD2 production in a mast cell degranulation assay and should be suitable to further explore H-PGDS biology.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Oxirredutases Intramoleculares/antagonistas & inibidores , Lipocalinas/antagonistas & inibidores , Quinolinas/química , Quinolinas/farmacologia , Animais , Descoberta de Drogas , Inibidores Enzimáticos/farmacocinética , Humanos , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/metabolismo , Lipocalinas/química , Lipocalinas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Quinolinas/farmacocinética
3.
Biometals ; 32(3): 453-467, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30810876

RESUMO

Salmonella enterica serovar Enteritidis (SE) is the most frequently-detected Salmonella in foodborne outbreaks in the European Union. Among such outbreaks, egg and egg products were identified as the most common vehicles of infection. Possibly, the major antibacterial property of egg white is iron restriction, which results from the presence of the iron-binding protein, ovotransferrin. To circumvent iron restriction, SE synthesise catecholate siderophores (i.e. enterobactin and salmochelin) that can chelate iron from host iron-binding proteins. Here, we highlight the role of lipocalin-like proteins found in egg white that could enhance egg-white iron restriction through sequestration of certain siderophores, including enterobactin. Indeed, it is now apparent that the egg-white lipocalin, Ex-FABP, can inhibit bacterial growth via its siderophore-binding capacity in vitro. However, it remains unclear whether Ex-FABP performs such a function in egg white or during bird infection. Regarding the two other lipocalins of egg white (Cal-γ and α-1-glycoprotein), there is currently no evidence to indicate that they sequester siderophores.


Assuntos
Antibacterianos/farmacologia , Clara de Ovo/química , Ferro/metabolismo , Lipocalinas/metabolismo , Salmonella enterica/efeitos dos fármacos , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Galinhas , Clara de Ovo/microbiologia , Lipocalinas/química , Testes de Sensibilidade Microbiana , Salmonella enterica/crescimento & desenvolvimento
4.
Int J Mol Med ; 43(3): 1531-1541, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30664181

RESUMO

Dogs are a major source of indoor allergens. However, the prevalence of dog allergies in China remains unclear, especially in children. In the present study, Can f 7, a canine allergen belonging to the Niemann pick type C2 protein family, was selected to study its sensitization rate in Chinese children with dog allergies. The Can f 7 gene was subcloned into a pET­28a vector and expressed in Escherichia coli BL21 (DE3) cells. Recombinant Can f 7 was purified by nickel affinity chromatography, identified by SDS­PAGE electrophoresis, and had its allergenicity assessed by western blot, ELISA and basophil activation tests. Through a series of bioinformatical approaches, B­cell epitopes, secondary structures, and 3 dimensional (3D) homology modeling of Can f 7 were predicted. The activity of the B cell epitopes was verified by ELISA. The recombinant Can f 7 showed a distinct band with a molecular weight of 14 kDa. Six of 20 sera from dog­allergic children reacted positively to the Can f 7. Can f 7 induced an ~4.0­fold increase in cluster of differentiation 63 and C­C motif chemokine receptor R3 expression in basophils sensitized with the serum of dog­allergic children compared with those of non­allergic controls. The secondary structure analysis showed that Can f 7 contains 6 ß­sheets. Five B cell epitopes of Can f 7 were predicted, and two of these were confirmed by ELISA. These results indicate that Can f 7 is an important canine allergen in Chinese children and provide novel data for further research concerning the use of Can f 7 in the diagnosis and treatment of Chinese children with canine allergy symptoms.


Assuntos
Alérgenos/genética , Alérgenos/imunologia , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Expressão Gênica , Lipocalinas/genética , Lipocalinas/imunologia , Adolescente , Alérgenos/química , Alérgenos/isolamento & purificação , Sequência de Aminoácidos , Animais , Criança , Pré-Escolar , Códon , Cães , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito B/química , Feminino , Humanos , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Lactente , Lipocalinas/química , Lipocalinas/isolamento & purificação , Masculino , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes
5.
Int J Mol Sci ; 19(12)2018 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-30486502

RESUMO

Fluorogens are an attractive type of dye for imaging applications, eliminating time-consuming washout steps from staining protocols. With just a handful of reported fluorogen-protein pairs, mostly in the green region of spectra, there is a need for the expansion of their spectral range. Still, the origins of solvatochromic and fluorogenic properties of the chromophores suitable for live-cell imaging are poorly understood. Here we report on the synthesis and labeling applications of novel red-shifted fluorogenic cell-permeable green fluorescent protein (GFP) chromophore analogs.


Assuntos
Proteínas de Fluorescência Verde/química , Lipocalinas/química , Microscopia de Fluorescência
6.
J Struct Biol ; 203(3): 205-218, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29885491

RESUMO

Apolipoprotein-D is a 25 kDa glycosylated member of the lipocalin family that folds into an eight-stranded ß-barrel with a single adjacent α-helix. Apolipoprotein-D specifically binds a range of small hydrophobic ligands such as progesterone and arachidonic acid and has an antioxidant function that is in part due to the reduction of peroxidised lipids by methionine-93. Therefore, apolipoprotein-D plays multiple roles throughout the body and is protective in Alzheimer's disease, where apolipoprotein-D overexpression reduces the amyloid-ß burden in Alzheimer's disease mouse models. Oligomerisation is a common feature of lipocalins that can influence ligand binding. The native structure of apolipoprotein-D, however, has not been conclusively defined. Apolipoprotein-D is generally described as a monomeric protein, although it dimerises when reducing peroxidised lipids. Here, we investigated the native structure of apolipoprotein-D derived from plasma, breast cyst fluid (BCF) and cerebrospinal fluid. In plasma and cerebrospinal fluid, apolipoprotein-D was present in high-molecular weight complexes, potentially in association with lipoproteins. In contrast, apolipoprotein-D in BCF formed distinct oligomeric species. We assessed apolipoprotein-D oligomerisation using native apolipoprotein-D purified from BCF and a suite of complementary methods, including multi-angle laser light scattering, analytical ultracentrifugation and small-angle X-ray scattering. Our analyses showed that apolipoprotein-D predominantly forms a ∼95 to ∼100 kDa tetramer. Small-angle X-ray scattering analysis confirmed these findings and provided a structural model for apolipoprotein-D tetramer. These data indicate apolipoprotein-D rarely exists as a free monomer under physiological conditions and provide insights into novel native structures of apolipoprotein-D and into oligomerisation behaviour in the lipocalin family.


Assuntos
Doença de Alzheimer/genética , Apolipoproteínas D/química , Conformação Proteica , Multimerização Proteica , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Animais , Apolipoproteínas D/líquido cefalorraquidiano , Apolipoproteínas D/genética , Cisto Mamário/química , Cristalografia por Raios X , Modelos Animais de Doenças , Humanos , Ligantes , Lipocalinas/química , Camundongos , Ligação Proteica , Espalhamento a Baixo Ângulo
7.
BioDrugs ; 32(3): 233-243, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29748739

RESUMO

Anticalin proteins are an emerging class of clinical-stage biopharmaceuticals with high potential as an alternative to antibodies. Anticalin molecules are generated by combinatorial design from natural lipocalins, which are abundant plasma proteins in humans, and reveal a simple, compact fold dominated by a central ß-barrel, supporting four structurally variable loops that form a binding site. Reshaping of this loop region results in Anticalin proteins that can recognize and tightly bind a wide range of medically relevant targets, from small molecules to peptides and proteins, as validated by X-ray structural analysis. Their robust format allows for modification in several ways, both as fusion proteins and by chemical conjugation, for example, to tune plasma half-life. Antagonistic Anticalin therapeutics have been developed for systemic administration (e.g., PRS-080: anti-hepcidin) or pulmonary delivery (e.g. PRS-060/AZD1402: anti-interleukin [IL]-4-Rα). Moreover, Anticalin proteins allow molecular formatting as bi- and even multispecific fusion proteins, especially in combination with antibodies that provide a second specificity. For example, PRS-343, which has recently entered clinical-stage development, combines an agonistic Anticalin targeting the costimulatory receptor 4-1BB with an antibody directed against the cancer antigen human epidermal growth factor receptor 2 (HER2), thus offering a novel treatment option in immuno-oncology.


Assuntos
Doença , Tratamento Farmacológico/métodos , Lipocalinas/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Biofarmácia , Humanos , Lipocalinas/química , Lipocalinas/genética , Terapia de Alvo Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Tecnologia Farmacêutica
8.
J Pharmacol Exp Ther ; 365(2): 368-378, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29463608

RESUMO

Since it was recently reported that an antibody for proprotein convertase subtilisin/kexin type 9 (PCSK9) reduces the risk of cardiovascular events in a clinical context, PCSK9 inhibition is thought to be an attractive therapy for dyslipidemia. In the present study, we created a novel small biologic alternative to PCSK9 antibodies called DS-9001a, comprising an albumin binding domain fused to an artificial lipocalin mutein (ABD-fused Anticalin protein), which can be produced by a microbial production system. DS-9001a strongly interfered with PCSK9 binding to low-density-lipoprotein receptor (LDL-R) and PCSK9-mediated degradation of LDL-R. In cynomolgus monkeys, single DS-9001a administration significantly reduced the serum LDL-C level up to 21 days (62.4% reduction at the maximum). Moreover, DS-9001a reduced plasma non-high-density-lipoprotein cholesterol and oxidized LDL levels, and their further reductions were observed when atorvastatin and DS-9001a were administered in combination in human cholesteryl ester transfer protein/ApoB double transgenic mice. Additionally, their reductions on the combination of atorvastatin and DS-9001a were more pronounced than those on the combination of atorvastatin and anacetrapib. Besides its favorable pharmacologic profile, DS-9001a has a lower molecular weight (about 22 kDa), yielding a high stoichiometric drug concentration that might result in a smaller administration volume than that in existing antibody therapy. Since bacterial production systems are viewed as more suited to mass production at low cost, DS-9001a may provide a new therapeutic option to treat patients with dyslipidemia. In addition, considering the growing demand for antibody-like drugs, ABD-fused Anticalin proteins could represent a promising new class of small biologic molecules.


Assuntos
Albuminas/metabolismo , Lipocalinas/genética , Pró-Proteína Convertase 9/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , Atorvastatina/farmacologia , Proteínas de Transferência de Ésteres de Colesterol , Interações Medicamentosas , Células Hep G2 , Humanos , Lipocalinas/química , Lipoproteínas LDL/sangue , Macaca fascicularis , Masculino , Camundongos , Oxazolidinonas/farmacologia , Domínios Proteicos , Ratos , Ratos Sprague-Dawley , Receptores de LDL/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
9.
J Comput Biol ; 25(3): 326-336, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29035579

RESUMO

Cryo-electron microscopy (cryo-EM) is a technique that produces three-dimensional density maps of large protein complexes. This allows for the study of the structure of these proteins. Identifying the secondary structures within proteins is vital to understanding the overall structure and function of the protein. The [Formula: see text]-barrel is one such secondary structure, commonly found in lipocalins and membrane proteins. In this article, we present a novel approach that utilizes genetic algorithms, kd-trees, and ray tracing to automatically detect and extract [Formula: see text]-barrels from cryo-EM density maps. This approach was tested on simulated and experimental density maps with zero, one, or multiple barrels in the density map. The results suggest that the proposed approach is capable of performing automatic detection of [Formula: see text]-barrels from medium resolution cryo-EM density maps.


Assuntos
Algoritmos , Motivos de Aminoácidos , Microscopia Crioeletrônica/métodos , Análise de Sequência de Proteína/métodos , Animais , Humanos , Lipocalinas/química , Lipocalinas/genética
10.
Insect Mol Biol ; 27(2): 177-187, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29164729

RESUMO

Lipocalins are low molecular weight membrane transporters that are abundantly expressed in the salivary glands and other tissues of ticks. In this study, we identified a lipocalin-like molecule, designated as otlip, from the soft ticks Ornithodoros turicata, the vector for the relapsing fever causing spirochete Borrelia turicatae. We noted that the expression of otlip was developmentally regulated, with adult ticks expressing significantly higher levels in comparison to the larvae or nymphal ticks. Expression of otlip was evident in both fed and unfed O. turicata ticks, with significantly increased expression in the salivary glands in comparison to the midgut or ovary tissues. High conservation of the biogenic amine-binding motif was evident in the deduced primary amino acid sequence of Otlip. Protein modelling of Otlip revealed conservation of most of the residues involved in binding histamine or serotonin ligand. In vitro assays demonstrated binding of recombinant Otlip with histamine. Furthermore, prediction of post-translational modifications revealed that Otlip contained phosphorylation and myristoylation sites. Taken together, our study not only provides evidence for the presence of a lipocalin-like molecule in O. turicata ticks but also suggests a role for this molecule in the salivary glands of this medically important vector.


Assuntos
Proteínas de Artrópodes/genética , Expressão Gênica , Histamina/metabolismo , Lipocalinas/genética , Ornithodoros/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Sequência de Bases , Feminino , Perfilação da Expressão Gênica , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Lipocalinas/química , Lipocalinas/metabolismo , Ninfa/genética , Ninfa/crescimento & desenvolvimento , Ninfa/metabolismo , Ornithodoros/crescimento & desenvolvimento , Ornithodoros/metabolismo , Filogenia , Glândulas Salivares/metabolismo , Alinhamento de Sequência
11.
Sci Rep ; 7(1): 16057, 2017 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-29167574

RESUMO

Two crystal structures of Japanin, an 18 kDa immune-modulatory lipocalin from the Brown Ear Tick (Rhipicephalus appendiculatus), have been determined at 2.2 and 2.4 Å resolution. In both crystal forms the protein is in complex with cholesterol, which sits in a closed pocket at the centre of the lipocalin barrel. Both crystal forms are dimers, which are also observed in solution. Molecular modelling suggests that previously-described members of a tick protein family bearing high sequence homology to Japanin are also likely to bind cholesterol or cholesterol derivatives.


Assuntos
Colesterol/metabolismo , Células Dendríticas/metabolismo , Rhipicephalus/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Lipocalinas/química , Lipocalinas/metabolismo , Estrutura Secundária de Proteína , Homologia Estrutural de Proteína
12.
ACS Synth Biol ; 6(12): 2241-2247, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-28937743

RESUMO

The molecular recognition of carbohydrates plays a fundamental role in many biological processes. However, the development of carbohydrate-binding reagents for biomedical research and use poses a challenge due to the generally poor affinity of proteins toward sugars in aqueous solution. Here, we describe the effective molecular recognition of pyranose monosaccharides (in particular, galactose and mannose) by a rationally designed protein receptor based on the human lipocalin scaffold (Anticalin). Complexation relies on reversible covalent cis-diol boronate diester formation with a genetically encoded l-boronophenylalanine (Bpa) residue which was incorporated as a non-natural amino acid at a sterically permissive position in the ligand pocket of the Anticalin, as confirmed by X-ray crystallography. Compared with the metal-ion and/or avidity-dependent oligovalent lectins that prevail in nature, our approach offers a novel and promising route to generate tight sugar-binding reagents both as research reagents and for biomedical applications.


Assuntos
Ácidos Borônicos/química , Galactose/química , Lipocalinas/química , Manose/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Lipocalinas/genética
13.
Mol Pharm ; 14(10): 3558-3567, 2017 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-28829147

RESUMO

Low water solubility of candidate drug compounds is a major problem in pharmaceutical research and development. We developed a novel drug delivery system (DDS) for poorly water-soluble drugs using lipocalin-type prostaglandin D synthase (L-PGDS), which belongs to the lipocalin superfamily and binds a large variety of hydrophobic molecules. In this study, we comprehensively evaluated the capability of L-PGDS to bind and solubilize various poorly water-soluble drugs using structure-based docking. Docking simulations of 2892 commercially available approved drugs indicated that L-PGDS shows higher binding affinities for various drugs compared with 2-hydroxypropyl-ß-cyclodextrin. Five drugs selected from the top 100 with the highest binding affinities for L-PGDS exhibited very low solubility in PBS (pH 7.4). However, in the presence of 1 mM L-PGDS, the apparent solubility of all drugs improved markedly, from 19.5- to 166-fold. Calorimetric experiments on two drugs, telmisartan and imatinib, revealed that L-PGDS forms a 1:2 complex with each drug, with dissociation constants of 0.4-40.0 µM. Kinetic simulations of drug dissolution with L-PGDS indicated that the difference in free energy change (ΔΔG) between the insoluble state and the L-PGDS-bound state are within the range from -10 to +5 kJ mol-1. The ΔΔG value is a critical factor in evaluating whether a poorly water-soluble drug can be solubilized by L-PGDS. Collectively, these results demonstrate that in silico docking is a promising approach for identifying drug molecules suitable for the L-PGDS-based DDS.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Liberação Controlada de Fármacos , Oxirredutases Intramoleculares/química , Lipocalinas/química , Simulação de Acoplamento Molecular , Benzimidazóis/química , Benzimidazóis/farmacocinética , Benzoatos/química , Benzoatos/farmacocinética , Calorimetria/métodos , Química Farmacêutica , Humanos , Mesilato de Imatinib/química , Mesilato de Imatinib/farmacocinética , Ligação Proteica , Proteínas Recombinantes/química , Solubilidade , Telmisartan , Água/química
14.
Biochem Biophys Res Commun ; 492(2): 166-171, 2017 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-28803983

RESUMO

Prostaglandins are involved in many physiological processes, and prostaglandin synthases facilitate the detoxification of xenobiotics as well as endogenous compounds, such as through glutathione conjugation. Specifically, prostaglandin D synthase (PGDS) catalyzes the isomerization of PGH2 to PGD2. Here we report the identification and structural analysis of PGDS from the brown planthopper rice pest Nilaparvata lugens (nlPGDS), which belongs to the sigma-class glutathione transferases. The structure of nlPGDS in complex with glutathione was determined at a resolution of 2.0 Å by X-ray crystallography. Bound glutathione was localized to the glutathione-binding site (G-site). Enzyme activity measurements following site-directed mutagenesis of nlPGDS indicated that amino acid residues Tyr8, Leu14, Trp39, Lys43, Gln50, Val51, Gln63, and Ser64 in the G-site contribute to its catalytic activity. To our knowledge, this represents the first report of a PGDS in insects. Our findings provide insights into the mechanism of nlPGDS activity and potentially that of other insects and therefore may facilitate the development of more effective and safe insecticides.


Assuntos
Glutationa/metabolismo , Hemípteros/enzimologia , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/metabolismo , Lipocalinas/química , Lipocalinas/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Hemípteros/química , Hemípteros/metabolismo , Modelos Moleculares , Oryza/parasitologia , Conformação Proteica
15.
Monoclon Antib Immunodiagn Immunother ; 36(4): 185-191, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28806153

RESUMO

Human lipocalin 6 (hLCN6) is a member of the lipocalin family, which is a group of structurally conserved hydrophobic ligand binding proteins, and widely distributed in animal, plant, and bacteria. Specific expression of hLCN6 in the epididymis and localization of this protein on the surface of spermatozoa suggest a role played by hLCN6, which may function as a transporter to carry ligands in the epididymal channel. However, the role of hLCN6 in sperm maturation has been largely unknown due to the lack of effective antibodies. In this study, we report the prokaryotic expression, purification, and refolding of recombinant hLCN6. Purified hLCN6 protein was used to generate monoclonal antibody (mAb) against this protein using conventional hybridoma techniques. The sensitivity and specificity of the anti-hLCN6 mAb were determined based on their activities in enzyme-linked immunosorbent assay and Western blotting analysis using various human tissues. The results showed that the antibody induced by recombinant hLCN6 protein had high sensitivity and specificity. Taken together, the recombinant hLCN6 protein and mAb against this protein obtained from our study provided useful tools for further exploration of the biological functions and molecular mechanism, as well as pathological significance of LCN6 in human.


Assuntos
Anticorpos Monoclonais Murinos/química , Lipocalinas/biossíntese , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/isolamento & purificação , Especificidade de Anticorpos , Western Blotting , Epididimo/metabolismo , Escherichia coli , Feminino , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Lipocalinas/química , Lipocalinas/imunologia , Masculino , Camundongos Endogâmicos BALB C , Redobramento de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia
16.
Sex Dev ; 11(3): 143-150, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28472794

RESUMO

Sex determination and sexual differentiation pathways are highly conserved between marsupials and eutherians. There are 2 different pathways of prostaglandin D2 (PGD2) synthesis: prostaglandin D synthase (PTGDS) and haematopoietic prostaglandin D synthase (HPGDS). PGD2 regulates the subcellular localization of SOX9 during gonadal sexual differentiation. To investigate the function of PGD2 in the tammar gonad, we cultured undifferentiated male gonads in the presence of the HPGDS inhibitor HQL-79 and female gonads with exogenous PGD2 to mimic activation of the PTGDS-PGD2 pathway. Tammar PTGDS and HPGDS have only 50% similarity with mouse and human orthologues, but functional domains are conserved. The expression of SOX9 was unchanged by the treatments in cultured gonads, but its subcellular localization was markedly affected. SOX9 remained cytoplasmic in the Sertoli cells of testes treated with HQL-79. Treated testes developed a thickened ovary-like surface epithelium. In contrast, SOX9 became nuclear in the granulosa cells of developing ovaries treated with PGD2 and the surface epithelium was thin, as in testes. These results demonstrate that PGD2 regulates the subcellular localization of SOX9 and subsequent gonadal development in the developing marsupial gonads, as it does in mice, and that it must have been an ancestral mechanism.


Assuntos
Núcleo Celular/metabolismo , Gônadas/metabolismo , Macropodidae/metabolismo , Prostaglandina D2/metabolismo , Fatores de Transcrição SOX9/metabolismo , Processos de Determinação Sexual , Sequência de Aminoácidos , Animais , Núcleo Celular/efeitos dos fármacos , Sequência Conservada , Feminino , Gônadas/efeitos dos fármacos , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/metabolismo , Lipocalinas/química , Lipocalinas/metabolismo , Masculino , Modelos Biológicos , Ovário/efeitos dos fármacos , Ovário/metabolismo , Piperidinas/farmacologia , Transporte Proteico/efeitos dos fármacos , Fatores de Transcrição SOX9/genética , Processos de Determinação Sexual/efeitos dos fármacos , Diferenciação Sexual/efeitos dos fármacos , Testículo/efeitos dos fármacos , Testículo/metabolismo
17.
Cleft Palate Craniofac J ; 54(4): 381-390, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-27243669

RESUMO

OBJECTIVE: Tinagl1 has a weak genetic association with craniosynostosis, but its functions in cartilage and bone development are unknown. Knockdown of Tinagl1 in zebrafish embryos allowed an initial characterization of its potential effects on craniofacial cartilage development and a test of whether these effects could involve Wnt signaling. RESULTS: Tinagl1 knockdown resulted in dose-dependent reductions and defects in ventral pharyngeal arch cartilages as well as the ethmoid plate, a zebrafish correlate to the palate. These defects could be correlated to reduced numbers of cranial neural crest cells in the pharyngeal arches and could be reproduced with comanipulation of Tinagl1 and Wnt3a by morpholino-based knockdown. CONCLUSIONS: These results suggest that Tinagl1 is required early in the proliferation or migration of cranial neural crest cells and that its effects are mediated via Wnt3a signaling. Because Wnt3a is among the Wnts that contribute to nonsyndromic cleft lip and cleft palate in mouse and man, further investigation of Tinagl1 may help to elucidate mechanisms underlying these disorders.


Assuntos
Região Branquial/anormalidades , Região Branquial/metabolismo , Cartilagem/anormalidades , Cartilagem/metabolismo , Anormalidades Craniofaciais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Lipocalinas/metabolismo , Proteína Wnt3A/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Movimento Celular , Proliferação de Células , Anormalidades Craniofaciais/genética , Embrião não Mamífero/metabolismo , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Hibridização In Situ , Lipocalinas/química , Lipocalinas/genética , Reação em Cadeia da Polimerase , Proteína Wnt3A/química , Proteína Wnt3A/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética
18.
J Biomed Mater Res A ; 105(3): 847-858, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27885823

RESUMO

For targeted brain delivery, nanoparticles (NPs) should bypass the blood-brain barrier (BBB). Novel functionalization strategies, based on low-density lipoprotein receptor (LDLR) binding domain, have been here tested to increase the brain targeting efficacy of poly d,l-lactic-co-glycolic acid (PLGA) NPs, biodegradable and suited for biomedical applications. Custom-made PLGA NPs were functionalized with an apolipoprotein E modified peptide (pep-apoE) responsible for LDLR binding, or with lipocalin-type prostaglandin-d-synthase (L-PGDS), highly expressed in the brain. At the comparison of pep-apoE and L-PGDS sequences, a highly homologs region was here identified, indicating that also L-PGDS could bind LDLR. Non-functionalized and functionalized NPs did not affect the viability of cultured human dendritic cells, protagonists of the immune response, and did not activate them to a proinflammatory profile. At 2 h after intravenous injection in mice, functionalized, but not the non-functionalized ones, fluorescent-tagged NPs were observed in the cerebral cortex parenchyma. The NPs were mostly internalized by neurons and microglia; glial cells showed a weak activation. The findings indicate that the tested functionalization strategies do not elicit adverse immune responses and that the peptidic moieties enable BBB traversal of the NPs, thus providing potential brain drug carriers. These could be especially effective for brain diseases in which LDLR is involved. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 847-858, 2017.


Assuntos
Barreira Hematoencefálica/metabolismo , Córtex Cerebral/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Oxirredutases Intramoleculares , Ácido Láctico , Lipocalinas , Nanopartículas , Peptídeos , Ácido Poliglicólico , Receptores de LDL/química , Apolipoproteínas E/química , Apolipoproteínas E/farmacocinética , Apolipoproteínas E/farmacologia , Feminino , Humanos , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/farmacocinética , Oxirredutases Intramoleculares/farmacologia , Ácido Láctico/química , Ácido Láctico/farmacocinética , Ácido Láctico/farmacologia , Lipocalinas/química , Lipocalinas/farmacocinética , Lipocalinas/farmacologia , Masculino , Nanopartículas/química , Nanopartículas/uso terapêutico , Peptídeos/química , Peptídeos/farmacocinética , Peptídeos/farmacologia , Ácido Poliglicólico/química , Ácido Poliglicólico/farmacocinética , Ácido Poliglicólico/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico
19.
Sci Rep ; 6: 35900, 2016 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-27782155

RESUMO

Transportation of pheromones bound with carrier proteins belonging to lipocalin superfamily is known to prolong chemo-signal communication between individuals belonging to the same species. Members of lipocalin family (MLF) proteins have three structurally conserved motifs for delivery of hydrophobic molecules to the specific recognizer. However, computational analyses are critically required to validate and emphasize the sequence and structural annotation of MLF. This study focused to elucidate the evolution, structural documentation, stability and binding efficiency of estrus urinary lipocalin protein (EULP) with endogenous pheromones adopting in-silico and fluorescence study. The results revealed that: (i) EULP perhaps originated from fatty acid binding protein (FABP) revealed in evolutionary analysis; (ii) Dynamic simulation study shows that EULP is highly stable at below 0.45 Å of root mean square deviation (RMSD); (iii) Docking evaluation shows that EULP has higher binding energy with farnesol and 2-iso-butyl-3-methoxypyrazine (IBMP) than 2-naphthol; and (iv) Competitive binding and quenching assay revealed that purified EULP has good binding interaction with farnesol. Both, In-silico and experimental studies showed that EULP is an efficient binding partner to pheromones. The present study provides impetus to create a point mutation for increasing longevity of EULP to develop pheromone trap for rodent pest management.


Assuntos
Estro/urina , Lipocalinas/urina , Sequência de Aminoácidos , Animais , Ligação Competitiva , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Ligantes , Lipocalinas/química , Lipocalinas/genética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Feromônios/metabolismo , Filogenia , Ligação Proteica , Mapas de Interação de Proteínas , Ratos , Espectrometria de Fluorescência
20.
Sci Rep ; 6: 32372, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27584086

RESUMO

Evolution has provided ticks with an arsenal of bioactive saliva molecules that counteract host defense mechanisms. This salivary pharmacopoeia enables blood-feeding while enabling pathogen transmission. High-throughput sequencing of tick salivary glands has thus become a major focus, revealing large expansion within protein encoding gene families. Among these are lipocalins, ubiquitous barrel-shaped proteins that sequester small, typically hydrophobic molecules. This study was initiated by mining the Ixodes ricinus salivary gland transcriptome for specific, uncharacterized lipocalins: three were identified. Differential expression of these I. ricinus lipocalins during feeding at distinct developmental stages and in response to Borrelia afzelii infection suggests a role in transmission of this Lyme disease spirochete. A phylogenetic analysis using 803 sequences places the three I. ricinus lipocalins with tick lipocalins that sequester monoamines, leukotrienes and fatty acids. Both structural analysis and biophysical simulations generated robust predictions showing these I. ricinus lipocalins have the potential to bind monoamines similar to other tick species previously reported. The multidisciplinary approach employed in this study characterized unique lipocalins that play a role in tick blood-feeding and transmission of the most important tick-borne pathogen in North America and Eurasia.


Assuntos
Grupo Borrelia Burgdorferi/fisiologia , Ixodes/metabolismo , Ixodes/microbiologia , Lipocalinas/metabolismo , Saliva/metabolismo , Análise de Variância , Animais , Sequência de Bases , Sítios de Ligação , Vetores de Doenças , Ixodes/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Ligantes , Lipocalinas/química , Lipocalinas/classificação , Doença de Lyme/microbiologia , Camundongos , Filogenia , Estrutura Terciária de Proteína
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