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1.
Int J Mol Med ; 45(3): 753-768, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31985023

RESUMO

Influenza viruses often pose a serious threat to animals and human health. In an attempt to explore the potential of herbal medicine as a treatment for influenza virus infection, eleutheroside B1, a coumarin compound extracted from herba sarcandrae, was identified, which exhibited antiviral and anti­inflammatory activities against influenza A virus. In this study, high­throughput RNA sequencing and isobaric tags for relative and absolute quantification (iTRAQ) assays were performed to determine alterations in the non­coding RNA (ncRNA) transcriptome and proteomics. Bioinformatics and target prediction analyses were used to decipher the potential roles of altered ncRNAs in the function of eleutheroside B1. Furthermore, long ncRNA (lncRNA) and mRNA co­expressing networks were constructed to analyze the biological functions by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The analysis of RNA sequencing data revealed that 5 differentially expressed ncRNAs were upregulated and 3 ncRNAs were downregulated in the A549 cells infected with A/PR8/34/H1N1, with or without eleutheroside B1 treatment (PR8+eleu and PR8, respectively). Nuclear paraspeckle assembly transcript 1 (NEAT1) was differentially expressed between the PR8 and A549 cell groups. GO and KEGG pathway analyses indicated that eleutheroside B1 took advantage of the host cell biological processes and molecular function for its antiviral and anti­inflammatory activities, as well as for regulating cytokine­cytokine receptor interaction in the immune system, consistent with previous findings. The results of the iTRAQ assays indicated that L antigen family member 3 (LAGE3) protein, essential for tRNA processing, tRNA metabolic processes and ncRNA processing, was downregulated in the PR8+eleu compared with the PR8 group. In the present study, these comprehensive, large­scale data analysis enhanced the understanding of multiple aspects of the transcriptome and proteomics that are involved in the antiviral and anti­inflammatory activities of eleutheroside B1. These findings demonstrate the potential of eleutheroside B1 for use in the prevention and treatment of influenza A virus­mediated infections.

2.
J Cell Mol Med ; 24(6): 3303-3313, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31965715

RESUMO

Osteolytic skeletal disorders are caused by an imbalance in the osteoclast and osteoblast function. Suppressing the differentiation and resorptive function of osteoclast is a key strategy for treating osteolytic diseases. Dracorhodin perchlorate (D.P), an active component from dragon blood resin, has been used for facilitating wound healing and anti-cancer treatments. In this study, we determined the effect of D.P on osteoclast differentiation and function. We have found that D.P inhibited RANKL-induced osteoclast formation and resorbed pits of hydroxyapatite-coated plate in a dose-dependent manner. D.P also disrupted the formation of intact actin-rich podosome structures in mature osteoclasts and inhibited osteoclast-specific gene and protein expressions. Further, D.P was able to suppress RANKL-activated JNK, NF-κB and Ca2+ signalling pathways and reduces the expression level of NFATc1 as well as the nucleus translocation of NFATc1. Overall, these results indicated a potential therapeutic effect of D.P on osteoclast-related conditions.

3.
Int Immunopharmacol ; 67: 186-193, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30553912

RESUMO

The aggressive phenotype displayed by fibroblast-like synoviocytes (FLSs) contributes to cartilage and bone destruction in rheumatoid arthritis (RA). Betulinic acid has been demonstrated to have a positive therapeutic effect on tumor, inflammation and immune disorder, however, the effects of betulinic acid on RA FLSs have not been verified. Therefore, in the present study, we observed the effect of betulinic acid on the migration and invasion of RA FLSs and explored its underlying signal mechanisms. Our results showed that betulinic acid treatment suppressed the migration, invasion and reorganization of the actin cytoskeleton of RA FLSs. In addition, we found that the mRNA expression of IL-1ß, IL-6, IL-8 and IL-17A were markedly down-regulated by treatment with betulinic acid in TNF-α-induced RA FLSs. To gain insight into the molecular mechanisms, we evaluated the effect of betulinic acid on NF-κB activation in RA FLSs. The results indicated that betulinic acid treatment reduced the TNF-α-induced activation of NF-κB signal pathway and the NF-κB nuclear accumulation. We also observed that treatment with betulinic acid attenuated synovial inflammation and joint destruction in mice with CIA. Taken together, these results suggest that betulinic acid inhibits the migration and invasion of RA FLSs by blocking NF-κB signal pathway activation.


Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Fibroblastos/efeitos dos fármacos , Inflamação/tratamento farmacológico , Sinoviócitos/efeitos dos fármacos , Triterpenos/uso terapêutico , Adulto , Idoso , Animais , Movimento Celular , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Fibroblastos/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos DBA , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Transdução de Sinais , Sinoviócitos/fisiologia
4.
EBioMedicine ; 39: 575-590, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30591370

RESUMO

BACKGROUND: Psoriasis is a common chronic inflammatory skin disease which lacks effective strategies for the treatment. Natural compounds with biological activities are good tools to identify new targets with therapeutic potentials. Acetyl-11-keto-ß-boswellic acid (AKBA) is the most bioactive ingredient of boswellic acids, a group of compounds with anti-inflammatory and anti-cancer properties. Target identification of AKBA and metabolomics analysis of psoriasis helped to elucidate the molecular mechanism underlying its effect, and provide new target(s) to treat the disease. METHODS: To explore the targets and molecular mechanism of AKBA, we performed affinity purification, metabolomics analysis of HaCaT cells treated with AKBA, and epidermis of imiquimod (IMQ) induced mouse model of psoriasis and psoriasis patients. FINDINGS: AKBA directly interacts with methionine adenosyltransferase 2A (MAT2A), inhibited its enzyme activity, decreased level of S-adenosylmethionine (SAM) and SAM/SAH ratio, and reprogrammed one­carbon metabolism in HaCaT cells. Untargeted metabolomics of epidermis showed one­carbon metabolism was activated in psoriasis patients. Topical use of AKBA improved inflammatory phenotype of IMQ induced psoriasis-like mouse model. Molecular docking and site-directed mutagenesis revealed AKBA bound to an allosteric site at the interface of MAT2A dimer. INTERPRETATION: Our study extends the molecular mechanism of AKBA by revealing a new interacting protein MAT2A. And this leads us to find out the dysregulated one­carbon metabolism in psoriasis, which indicates the therapeutic potential of AKBA in psoriasis. FUND: The National Natural Science Foundation, the National Program on Key Basic Research Project, the Shanghai Municipal Commission, the Leading Academic Discipline Project of the Shanghai Municipal Education Commission.


Assuntos
Carbono/metabolismo , Metabolômica/métodos , Metionina Adenosiltransferase/antagonistas & inibidores , Psoríase/tratamento farmacológico , Triterpenos/administração & dosagem , Administração Tópica , Sítio Alostérico/efeitos dos fármacos , Animais , Linhagem Celular , Regulação para Baixo , Humanos , Imiquimode/efeitos adversos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Masculino , Metionina Adenosiltransferase/química , Camundongos , Modelos Moleculares , Simulação de Acoplamento Molecular , Conformação Proteica , Psoríase/induzido quimicamente , Psoríase/metabolismo , Triterpenos/farmacologia
5.
Int Immunopharmacol ; 55: 174-182, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29268189

RESUMO

In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLSs) play an essential role in cartilage destruction. Aggressive migration and invasion by FLSs significantly affect RA pathology. Kaempferol has been shown to inhibit cancer cell migration and invasion. However, the effects of kaempferol on RA FLSs have not been investigated. Our study aimed to determine the effects of kaempferol on RA both in vitro and in vivo. In vitro, cell migration and invasion were measured using scratch assays and the Boyden chamber method, respectively. The cytoskeletal reorganization of RA FLSs was evaluated by immunofluorescence staining. Matrix metalloproteinase (MMP) levels were measured by real-time PCR, and protein expression levels were measured by western blotting. In vivo, the effects of kaempferol were evaluated in mice with CIA. The results showed that kaempferol reduced migration, invasion and MMP expression in RA FLSs. In addition, we demonstrated that kaempferol inhibited reorganization of the actin cytoskeleton during cell migration. Moreover, kaempferol dramatically suppressed tumor necrosis factor (TNF)-α-induced MAPK activation without affecting the expression of TNF-α receptors. We also demonstrated that kaempferol attenuated the severity of arthritis in mice with CIA. Taken together, these results suggested that kaempferol inhibits the migration and invasion of FLSs in RA by blocking MAPK pathway activation without affecting the expression of TNF-α receptors.


Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Fibroblastos/fisiologia , Quempferóis/uso terapêutico , Sinoviócitos/fisiologia , Actinas/metabolismo , Adulto , Idoso , Animais , Artrite Experimental , Movimento Celular , Células Cultivadas , Citoesqueleto/metabolismo , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos DBA , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/metabolismo
6.
J Biol Chem ; 291(48): 24900-24911, 2016 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-27681598

RESUMO

Lamprey angiotensinogen (l-ANT) is a hormone carrier in the regulation of blood pressure, but it is also a heparin-dependent thrombin inhibitor in lamprey blood coagulation system. The detailed mechanisms on how angiotensin is carried by l-ANT and how heparin binds l-ANT and mediates thrombin inhibition are unclear. Here we have solved the crystal structure of cleaved l-ANT at 2.7 Šresolution and characterized its properties in heparin binding and protease inhibition. The structure reveals that l-ANT has a conserved serpin fold with a labile N-terminal angiotensin peptide and undergoes a typical stressed-to-relaxed conformational change when the reactive center loop is cleaved. Heparin binds l-ANT tightly with a dissociation constant of ∼10 nm involving ∼8 monosaccharides and ∼6 ionic interactions. The heparin binding site is located in an extensive positively charged surface area around helix D involving residues Lys-148, Lys-151, Arg-155, and Arg-380. Although l-ANT by itself is a poor thrombin inhibitor with a second order rate constant of 500 m-1 s-1, its interaction with thrombin is accelerated 90-fold by high molecular weight heparin following a bell-shaped dose-dependent curve. Short heparin chains of 6-20 monosaccharide units are insufficient to promote thrombin inhibition. Furthermore, an l-ANT mutant with the P1 Ile mutated to Arg inhibits thrombin nearly 1500-fold faster than the wild type, which is further accelerated by high molecular weight heparin. Taken together, these results suggest that heparin binds l-ANT at a conserved heparin binding site around helix D and promotes the interaction between l-ANT and thrombin through a template mechanism conserved in vertebrates.


Assuntos
Angiotensinas/química , Proteínas de Peixes/química , Heparina/química , Lampreias , Trombina/química , Angiotensinas/genética , Angiotensinas/metabolismo , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Heparina/genética , Heparina/metabolismo , Mutação , Domínios Proteicos , Estrutura Secundária de Proteína , Trombina/genética , Trombina/metabolismo
7.
J Biol Chem ; 291(30): 15674-86, 2016 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-27246852

RESUMO

The Z mutation (E342K) of α1-antitrypsin (α1-AT), carried by 4% of Northern Europeans, predisposes to early onset of emphysema due to decreased functional α1-AT in the lung and to liver cirrhosis due to accumulation of polymers in hepatocytes. However, it remains unclear why the Z mutation causes intracellular polymerization of nascent Z α1-AT and why 15% of the expressed Z α1-AT is secreted into circulation as functional, but polymerogenic, monomers. Here, we solve the crystal structure of the Z-monomer and have engineered replacements to assess the conformational role of residue Glu-342 in α1-AT. The results reveal that Z α1-AT has a labile strand 5 of the central ß-sheet A (s5A) with a consequent equilibrium between a native inhibitory conformation, as in its crystal structure here, and an aberrant conformation with s5A only partially incorporated into the central ß-sheet. This aberrant conformation, induced by the loss of interactions from the Glu-342 side chain, explains why Z α1-AT is prone to polymerization and readily binds to a 6-mer peptide, and it supports that annealing of s5A into the central ß-sheet is a crucial step in the serpins' metastable conformational formation. The demonstration that the aberrant conformation can be rectified through stabilization of the labile s5A by binding of a small molecule opens a potential therapeutic approach for Z α1-AT deficiency.


Assuntos
Mutação de Sentido Incorreto , Deficiência de alfa 1-Antitripsina , alfa 1-Antitripsina/química , Substituição de Aminoácidos , Cristalografia por Raios X , Humanos , Estabilidade Proteica , Estrutura Secundária de Proteína , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo
8.
J Biol Chem ; 291(6): 2954-66, 2016 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-26631724

RESUMO

BMP10 is highly expressed in the developing heart and plays essential roles in cardiogenesis. BMP10 deletion in mice results in embryonic lethality because of impaired cardiac development. In adults, BMP10 expression is restricted to the right atrium, though ventricular hypertrophy is accompanied by increased BMP10 expression in a rat hypertension model. However, reports of BMP10 activity in the circulation are inconclusive. In particular, it is not known whether in vivo secreted BMP10 is active or whether additional factors are required to achieve its bioactivity. It has been shown that high-affinity binding of the BMP10 prodomain to the mature ligand inhibits BMP10 signaling activity in C2C12 cells, and it was proposed that prodomain-bound BMP10 (pBMP10) complex is latent. In this study, we demonstrated that the BMP10 prodomain did not inhibit BMP10 signaling activity in multiple endothelial cells, and that recombinant human pBMP10 complex, expressed in mammalian cells and purified under native conditions, was fully active. In addition, both BMP10 in human plasma and BMP10 secreted from the mouse right atrium were fully active. Finally, we confirmed that active BMP10 secreted from mouse right atrium was in the prodomain-bound form. Our data suggest that circulating BMP10 in adults is fully active and that the reported vascular quiescence function of BMP10 in vivo is due to the direct activity of pBMP10 and does not require an additional activation step. Moreover, being an active ligand, recombinant pBMP10 may have therapeutic potential as an endothelial-selective BMP ligand, in conditions characterized by loss of BMP9/10 signaling.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Cardiomegalia/metabolismo , Células Endoteliais/metabolismo , Transdução de Sinais , Animais , Proteínas Morfogenéticas Ósseas/genética , Cardiomegalia/genética , Cardiomegalia/patologia , Linhagem Celular , Células Endoteliais/patologia , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Humanos , Camundongos , Ratos
9.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 9): 1135-8, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26323298

RESUMO

Kallistatin is a serine protease inhibitor (serpin) which specifically inhibits human tissue kallikrein; however, its inhibitory activity is inhibited by heparin. In order to elucidate the underlying mechanism, recombinant human kallistatin was prepared in Escherichia coli and the protein was crystallized by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 1.9 Šresolution. The crystals were found to belong to space group P61, with unit-cell parameters a = 113.51, b = 113.51, c = 76.17 Å. Initial analysis indicated that the crystallized kallistatin was in a relaxed conformation, with its reactive-centre loop inserted in the central ß-sheet.


Assuntos
Serpinas/química , Cromatografia por Troca Iônica , Cristalização , Cristalografia por Raios X , Humanos , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Serpinas/isolamento & purificação , Eletricidade Estática
10.
World J Gastroenterol ; 21(8): 2425-32, 2015 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-25741151

RESUMO

AIM: To report the incidence and potential risk factors of small-volume chylous ascites (SVCA) following laparoscopic radical gastrectomy (LAG). METHODS: A total of 1366 consecutive gastric cancer patients who underwent LAG from January 2008 to June 2011 were enrolled in this study. We analyzed the patients based on the presence or absence of SVCA. RESULTS: SVCA was detected in 57 (4.17%) patients, as determined by the small-volume drainage (range, 30-100 mL/24 h) of triglyceride-rich fluid. Both univariate and multivariate analyses revealed that the total number of resected lymph nodes (LNs), No. 8 or No. 9 LN metastasis and N stage were independent risk factors for SVCA following LAG (P<0.05). Regarding hospital stay, there was a significant difference between the groups with and without SVCA (P<0.001). The 3-year disease-free and overall survival rates of the patients with SVCA were 47.4% and 56.1%, respectively, which were similar to those of the patients without SVCA (P>0.05). CONCLUSION: SVCA following LAG developed significantly more frequently in the patients with ≥32 harvested LNs, ≥3 metastatic LNs, or No. 8 or No. 9 LN metastasis. SVCA, which was successfully treated with conservative management, was associated with a prolonged hospital stay but was not associated with the prognosis.


Assuntos
Ascite Quilosa/epidemiologia , Gastrectomia/efeitos adversos , Laparoscopia/efeitos adversos , Neoplasias Gástricas/cirurgia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Distribuição de Qui-Quadrado , Criança , China/epidemiologia , Ascite Quilosa/diagnóstico , Bases de Dados Factuais , Intervalo Livre de Doença , Feminino , Gastrectomia/métodos , Gastrectomia/mortalidade , Humanos , Incidência , Estimativa de Kaplan-Meier , Laparoscopia/métodos , Laparoscopia/mortalidade , Tempo de Internação , Modelos Logísticos , Excisão de Linfonodo/efeitos adversos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estudos Retrospectivos , Fatores de Risco , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Fatores de Tempo , Resultado do Tratamento , Adulto Jovem
11.
J Biol Chem ; 289(45): 31150-9, 2014 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-25237187

RESUMO

BMP9, a member of the TGFß superfamily, is a homodimer that forms a signaling complex with two type I and two type II receptors. Signaling through high-affinity activin receptor-like kinase 1 (ALK1) in endothelial cells, circulating BMP9 acts as a vascular quiescence factor, maintaining endothelial homeostasis. BMP9 is also the most potent BMP for inducing osteogenic signaling in mesenchymal stem cells in vitro and promoting bone formation in vivo. This activity requires ALK1, the lower affinity type I receptor ALK2, and higher concentrations of BMP9. In adults, BMP9 is constitutively expressed in hepatocytes and secreted into the circulation. Optimum concentrations of BMP9 are essential to maintain the highly specific endothelial-protective function. Factors regulating BMP9 stability and activity remain unknown. Here, we showed by chromatography and a 1.9 Å crystal structure that stable BMP9 dimers could form either with (D-form) or without (M-form) an intermolecular disulfide bond. Although both forms of BMP9 were capable of binding to the prodomain and ALK1, the M-form demonstrated less sustained induction of Smad1/5/8 phosphorylation. The two forms could be converted into each other by changing the redox potential, and this redox switch caused a major alteration in BMP9 stability. The M-form displayed greater susceptibility to redox-dependent cleavage by proteases present in serum. This study provides a mechanism for the regulation of circulating BMP9 concentrations and may provide new rationales for approaches to modify BMP9 levels for therapeutic purposes.


Assuntos
Regulação da Expressão Gênica , Fatores de Diferenciação de Crescimento/metabolismo , Oxirredução , Animais , Linhagem Celular , Cristalização , Cristalografia por Raios X , DNA Complementar/metabolismo , Dimerização , Dissulfetos/química , Células Endoteliais/citologia , Fator 2 de Diferenciação de Crescimento/metabolismo , Humanos , Ligantes , Camundongos , Fenótipo , Proteólise , Proteínas Recombinantes/metabolismo , Transdução de Sinais
12.
J Biol Chem ; 286(18): 16163-73, 2011 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-21325280

RESUMO

The release of hormones from thyroxine-binding globulin (TBG) and corticosteroid-binding globulin (CBG) is regulated by movement of the reactive center loop in and out of the ß-sheet A of the molecule. To investigate how these changes are transmitted to the hormone-binding site, we developed a sensitive assay using a synthesized thyroxine fluorophore and solved the crystal structures of reactive loop cleaved TBG together with its complexes with thyroxine, the thyroxine fluorophores, furosemide, and mefenamic acid. Cleavage of the reactive loop results in its complete insertion into the ß-sheet A and a substantial but incomplete decrease in binding affinity in both TBG and CBG. We show here that the direct interaction between residue Thr(342) of the reactive loop and Tyr(241) of the hormone binding site contributes to thyroxine binding and release following reactive loop insertion. However, a much larger effect occurs allosterically due to stretching of the connecting loop to the top of the D helix (hD), as confirmed in TBG with shortening of the loop by three residues, making it insensitive to the S-to-R transition. The transmission of the changes in the hD loop to the binding pocket is seen to involve coherent movements in the s2/3B loop linked to the hD loop by Lys(243), which is, in turn, linked to the s4/5B loop, flanking the thyroxine-binding site, by Arg(378). Overall, the coordinated movements of the reactive loop, hD, and the hormone binding site allow the allosteric regulation of hormone release, as with the modulation demonstrated here in response to changes in temperature.


Assuntos
Corticosteroides/química , Globulina de Ligação a Tiroxina/química , Tiroxina/química , Transcortina/química , Corticosteroides/genética , Corticosteroides/metabolismo , Regulação Alostérica/fisiologia , Sítios de Ligação , Humanos , Estrutura Secundária de Proteína , Tiroxina/genética , Tiroxina/metabolismo , Globulina de Ligação a Tiroxina/genética , Globulina de Ligação a Tiroxina/metabolismo , Transcortina/genética , Transcortina/metabolismo
13.
Nature ; 468(7320): 108-11, 2010 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-20927107

RESUMO

Blood pressure is critically controlled by angiotensins, which are vasopressor peptides specifically released by the enzyme renin from the tail of angiotensinogen-a non-inhibitory member of the serpin family of protease inhibitors. Although angiotensinogen has long been regarded as a passive substrate, the crystal structures solved here to 2.1 Å resolution show that the angiotensin cleavage site is inaccessibly buried in its amino-terminal tail. The conformational rearrangement that makes this site accessible for proteolysis is revealed in our 4.4 Å structure of the complex of human angiotensinogen with renin. The co-ordinated changes involved are seen to be critically linked by a conserved but labile disulphide bridge. Here we show that the reduced unbridged form of angiotensinogen is present in the circulation in a near 40:60 ratio with the oxidized sulphydryl-bridged form, which preferentially interacts with receptor-bound renin. We propose that this redox-responsive transition of angiotensinogen to a form that will more effectively release angiotensin at a cellular level contributes to the modulation of blood pressure. Specifically, we demonstrate the oxidative switch of angiotensinogen to its more active sulphydryl-bridged form in the maternal circulation in pre-eclampsia-the hypertensive crisis of pregnancy that threatens the health and survival of both mother and child.


Assuntos
Angiotensinogênio/química , Angiotensinogênio/metabolismo , Angiotensinas/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Angiotensinogênio/sangue , Angiotensinas/química , Pressão Sanguínea , Cristalografia por Raios X , Dissulfetos/química , Dissulfetos/metabolismo , Feminino , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Estresse Oxidativo , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/metabolismo , Gravidez , Conformação Proteica , Renina/química , Renina/metabolismo
14.
Mol Biosyst ; 5(9): 1025-31, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19668868

RESUMO

Plasminogen activator inhibitor-1 (PAI-1) is a member of the serpin (serine protease inhibitor) superfamily. Like most serpins, the inhibitory function of PAI-1 relies on a flexible reactive centre loop (RCL) undertaking a striking conformational transition. We have investigated the conformational dynamics of the RCL of PAI-1 by time-resolved fluorescence anisotropy. A heterogeneous population model with three rotational correlation times has been employed to account for the "dip and rise" observed in fluorescence anisotropy decay curves. The RCL becomes almost fully solvent exposed and exhibits faster rotation when PAI-1 interacts with a RCL-mimicking octapeptide which blocks the loop insertion pathway, indicating that the RCL is well displaced from the protein surface; while the binding of Somatomedin B (SMB) domain of vitronectin, only induces small changes in the RCL. Comparison of the fluorescence lifetime and anisotropy decay of the wild-type PAI-1 with that of the stabilised mutant suggests that there would be no major structural differences between them. Our results indicate that in a native serpin, the P14 residue of the hinge region can flip in and out of the central beta-sheet A more readily than previously thought, which is likely an inherent property for serpins' protease inhibitory function.


Assuntos
Polarização de Fluorescência/métodos , Nanotecnologia/métodos , Inibidor 1 de Ativador de Plasminogênio/química , Corantes Fluorescentes/química , Modelos Moleculares , Naftalenossulfonatos/química , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes , Somatomedinas/metabolismo , Vitronectina/metabolismo
15.
Blood ; 114(17): 3662-7, 2009 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-19528533

RESUMO

Protein Z (PZ) binds to PZ-dependent inhibitor (ZPI) and accelerates the inhibition of the coagulation protease, activated factor X (FXa), in the presence of phospholipids and Ca2+. A 2.3A resolution crystal structure of PZ complexed with ZPI shows that ZPI is a typical serine protease inhibitor and that PZ has a serine protease fold with distorted oxyanion hole and S1 pocket. The 2 molecules bind with fully complementary surfaces spanning over 2400A(2) and involving extensive ionic and hydrophobic interactions. ZPI has an unusual shutter region with a negatively charged residue buried within the hydrophobic core of the molecule. This unique Asp(213) is critical in maintaining the balanced metastability required for optimal protease inhibition, especially when PZ is bound, with its replacement with Asn resulting in increased thermal stability, but decreased efficiency of protease inhibition. The structure of ZPI shows negatively and positively charged surfaces on top of the molecule, in keeping with mutagenesis studies in this work indicating exosite interactions with FXa when it docks on top of ZPI. As modeled in this study, the gamma-carboxy-glutamic acid-containing domains of PZ and FXa enable them to bind to the same phospholipid surfaces on platelet and other membranes, with optimal proximity for the inhibition of FXa by the complexed ZPI.


Assuntos
Proteínas Sanguíneas/química , Fator X/antagonistas & inibidores , Membranas/metabolismo , Serpinas/química , Sítio Alostérico , Sítios de Ligação , Coagulação Sanguínea , Cálcio/metabolismo , Dicroísmo Circular , Cristalização , Cristalografia por Raios X , Fator Xa/metabolismo , Humanos , Fosfolipídeos/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química
16.
J Mol Biol ; 380(1): 244-51, 2008 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-18513745

RESUMO

Corticosteroids are transported in the blood by a serpin, corticosteroid-binding globulin (CBG), and their normally equilibrated release can be further triggered by the cleavage of the reactive loop of CBG. We report here the crystal structures of cleaved human CBG (cCBG) at 1.8-A resolution and its complex with cortisol at 2.3-A resolution. As expected, on cleavage, CBG undergoes the irreversible S-to-R serpin transition, with the cleaved reactive loops being fully incorporated into the central beta-sheet. A connecting loop of helix D, which is in a helix-like conformation in native CBG, unwinds and grossly perturbs the hormone binding site following beta-sheet expansion in the cCBG structure but shifts away from the binding site by more than 8 A following the binding of cortisol. Unexpectedly, on cortisol binding, the hormone binding site of cCBG adopts a configuration almost identical with that of the native conformer. We conclude that CBG has adapted an allosteric mechanism of the serpins to allow equilibrated release of the hormones by a flip-flop movement of the intact reactive loop into and out of the beta-sheet. The change in the hormone binding affinity results from a change in the flexibility or plasticity of the connecting loop, which modulates the configuration of the binding site.


Assuntos
Corticosteroides/metabolismo , Transcortina/química , Regulação Alostérica , Cristalografia por Raios X , Humanos , Estrutura Secundária de Proteína , Proteínas de Ligação a Tiroxina/química
17.
Proc Natl Acad Sci U S A ; 103(36): 13321-6, 2006 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-16938877

RESUMO

The hormones that most directly control tissue activities in health and disease are delivered by two noninhibitory members of the serpin family of protease inhibitors, thyroxine-binding globulin (TBG) and corticosteroid-binding globulin. The structure of TBG bound to tetra-iodo thyroxine, solved here at 2.8 A, shows how the thyroxine is carried in a surface pocket on the molecule. This unexpected binding site is confirmed by mutations associated with a loss of hormone binding in both TBG and also homologously in corticosteroid-binding globulin. TBG strikingly differs from other serpins in having the upper half of its main beta-sheet fully opened, so its reactive center peptide loop can readily move in and out of the sheet to give an equilibrated binding and release of thyroxine. The entry of the loop triggers a conformational change, with a linked contraction of the binding pocket and release of the bound thyroxine. The ready reversibility of this change is due to the unique presence in the reactive loop of TBG of a proline that impedes the full and irreversible entry of the loop that occurs in other serpins. Thus, TBG has adapted the serpin inhibitory mechanism to give a reversible flip-flop transition, from a high-affinity to a low-affinity form. The complexity and ready triggering of this conformational mechanism strongly indicates that TBG has evolved to allow a modulated and targeted delivery of thyroxine to the tissues.


Assuntos
Proteínas de Ligação a Tiroxina/química , Proteínas de Ligação a Tiroxina/metabolismo , Tiroxina/sangue , Tiroxina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/genética , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Treonina/química , Treonina/metabolismo , Proteínas de Ligação a Tiroxina/genética , Proteínas de Ligação a Tiroxina/isolamento & purificação
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