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1.
Acta Crystallogr F Struct Biol Commun ; 74(Pt 11): 696-703, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30387774

RESUMO

Ectonucleotide phosphodiesterase/pyrophosphatase-3 (NPP3, ENPP3) is an ATP-hydrolyzing glycoprotein that is located in the extracellular space. The full-length ectodomain of rat NPP3 was expressed in HEK293S GntI- cells, purified using two chromatographic steps and crystallized. Its structure at 2.77 Šresolution reveals that the active-site zinc ions are missing and a large part of the active site and the surrounding residues are flexible. The SMB-like domains have the same orientation in all four molecules in the asymmetric unit. The SMB2 domain is oriented as in NPP2, but the SMB1 domain does not interact with the PDE domain but extends further away from the PDE domain. Deletion of the SMB domains resulted in crystals that diffracted to 2.4 Šresolution and are suitable for substrate-binding studies.


Assuntos
Diester Fosfórico Hidrolases/química , Pirofosfatases/química , Sítios de Ligação , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Células HEK293 , Humanos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Domínios Proteicos , Pirofosfatases/genética , Pirofosfatases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Somatomedinas/química
2.
Carbohydr Res ; 443-444: 29-36, 2017 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-28324771

RESUMO

Carbohydrate mimics have been studied for a long time as useful sugar substitutes, both in the investigation of biological events and in the treatment of sugar-related diseases. Here we report further evaluation of the capabilities of inositols as carbohydrate substitutes. The conformational features of an inositol-model of a simplified repeating unit corresponding to the capsular polysaccharide of Streptococcus pneumoniae 19F has been evaluated by computational analysis, and compared to the native repeating unit. The inositol mimic was synthesized, and its experimental spectroscopic data allowed for verification of the theoretical results.


Assuntos
Cápsulas Bacterianas/química , Inositol/química , Inositol/síntese química , Modelos Moleculares , Somatomedinas/química , Somatomedinas/síntese química , Streptococcus pneumoniae/química , Configuração de Carboidratos , Técnicas de Química Sintética
3.
PLoS One ; 11(12): e0168874, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28002497

RESUMO

Since the insulin-like growth factor 3 (igf3) gene was recently discovered in fish ovary, its function in the gonads has received much attention. In this study, we isolated two igf3 subtypes from common carp (Cyprinus carpio), which comprised full-length cDNA of 707 and 1153 nucleotides encoding 205 and 198 amino acids (aa), respectively. The Igf3 aa sequence had the highest gene homology of 72% with the corresponding sequence in zebrafish (Danio rerio). Phylogenetic tree construction revealed that the C. carpio igf3 gene was first clustered with D. rerio and then with other teleost species. Igf3 mRNA was widely expressed, with expression being highest in the gonads and blood. In the gonad development stage, igf3a mRNA expression was highest in the maturity and recession stage of the ovary, and decline phase of the testis, while igf3b was highest in the recession and fully mature periods of the ovaries and testes, respectively. Western blotting of testis protein samples showed two bands of approximately 21 kDa and 34 kDa corresponding to the calculated molecular mass of the two Igf3 subtypes; no signal was detected in the ovary. The Igf3 protein was localized in the ovary granulosa cells and testis spermatogonium and spermatids. 17ß-Ethinylestradiol treatment increased both ovary and testis igf3 mRNA expression. These findings suggest that Igf3 may play an important role in C. carpio gonadal development.


Assuntos
Carpas/genética , Proteínas de Peixes/genética , Gônadas/metabolismo , Somatomedinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Carpas/classificação , Carpas/metabolismo , Estradiol/farmacologia , Estrogênios/farmacologia , Feminino , Proteínas de Peixes/química , Proteínas de Peixes/classificação , Proteínas de Peixes/metabolismo , Gônadas/efeitos dos fármacos , Gônadas/crescimento & desenvolvimento , Masculino , Ovário/efeitos dos fármacos , Ovário/metabolismo , Ovário/patologia , Filogenia , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Somatomedinas/química , Somatomedinas/classificação , Somatomedinas/metabolismo , Testículo/efeitos dos fármacos , Testículo/metabolismo , Testículo/patologia , Transcriptoma/efeitos dos fármacos , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética
4.
Biochemistry ; 55(31): 4386-98, 2016 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-27416303

RESUMO

Plasminogen activator inhibitor type 1 (PAI-1) regulates the fibrinolysis pathway by inhibiting the protease activity of plasminogen activators. PAI-1 works in concert with vitronectin (VN), an extracellular protein that aids in localization of active PAI-1 to tissues. The Peterson laboratory demonstrated that Cu(II) and other transition metals modulate the stability of PAI-1, exhibiting effects that are dependent on the presence or absence of the somatomedin B (SMB) domain of VN. The study presented here dissects the changes in molecular dynamics underlying the destabilizing effects of Cu(II) on PAI-1. We utilize backbone amide hydrogen/deuterium exchange monitored by mass spectrometry to assess PAI-1 dynamics in the presence and absence of Cu(II) ions with and without the SMB domain of VN. We show that Cu(II) produces an increase in dynamics in regions important for the function and overall stability of PAI-1, while the SMB domain elicits virtually the opposite effect. A mutant form of PAI-1 lacking two N-terminal histidine residues at positions 2 and 3 exhibits similar increases in dynamics upon Cu(II) binding compared to that of active wild-type PAI-1, indicating that the observed structural effects are not a result of coordination of Cu(II) to these histidine residues. Finally, addition of Cu(II) results in an acceleration of the local unfolding kinetics of PAI-1 presumed to be on pathway to the latency conversion. The effect of ligands on the dynamics of PAI-1 adds another intriguing dimension to the mechanisms for regulation of PAI-1 stability and function.


Assuntos
Cobre/metabolismo , Inibidor 1 de Ativador de Plasminogênio/química , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Medição da Troca de Deutério/métodos , Fibrinólise , Histidina/química , Humanos , Cinética , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Somatomedinas/química , Somatomedinas/metabolismo , Resposta a Proteínas não Dobradas , Vitronectina/química , Vitronectina/metabolismo
5.
Mol Cancer Ther ; 15(7): 1602-13, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27196774

RESUMO

We provide proof-of-concept evidence for a new class of therapeutics that target growth factor:extracellular matrix (GF:ECM) interactions for the management of breast cancer. Insulin-like growth factor-I (IGF-I) forms multiprotein complexes with IGF-binding proteins (IGFBP) and the ECM protein vitronectin (VN), and stimulates the survival, migration and invasion of breast cancer cells. For the first time we provide physical evidence for IGFBP-3:VN interactions in breast cancer patient tissues; these interactions were predominantly localized to tumor cell clusters and in stroma surrounding tumor cells. We show that disruption of IGF-I:IGFBP:VN complexes with L(27)-IGF-II inhibits IGF-I:IGFBP:VN-stimulated breast cancer cell migration and proliferation in two- and three-dimensional assay systems. Peptide arrays screened to identify regions critical for the IGFBP-3/-5:VN and IGF-II:VN interactions demonstrated IGFBP-3/-5 and IGF-II binds VN through the hemopexin-2 domain, and VN binds IGFBP-3 at residues not involved in the binding of IGF-I to IGFBP-3. IGFBP-interacting VN peptides identified from these peptide arrays disrupted the IGF-I:IGFBP:VN complex, impeded the growth of primary tumor-like spheroids and, more importantly, inhibited the invasion of metastatic breast cancer cells in 3D assay systems. These studies provide first-in-field evidence for the utility of small peptides in antagonizing GF:ECM-mediated biologic functions and present data demonstrating the potential of these peptide antagonists as novel therapeutics. Mol Cancer Ther; 15(7); 1602-13. ©2016 AACR.


Assuntos
Neoplasias da Mama/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Somatomedinas/metabolismo , Vitronectina/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Imuno-Histoquímica , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/química , Ligantes , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Somatomedinas/química , Vitronectina/química
6.
J Pept Sci ; 22(5): 260-70, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26910514

RESUMO

The synthesis of insulin has inspired fundamental advances in the art of peptide science while simultaneously revealing the structure-function relationship of this centrally important metabolic hormone. This review highlights milestones in the chemical synthesis of insulin that can be divided into two separate approaches: (i) disulfide bond formation driven by protein folding and (ii) chemical reactivity-directed sequential disulfide bond formation. Common to the two approaches are the persistent challenges presented by the hydrophobic nature of the individual A-chain and B-chain and the need for selective disulfide formation under mildly oxidative conditions. The extension and elaboration of these synthetic approaches have been ongoing within the broader insulin superfamily. These structurally similar peptides include the insulin-like growth factors and also the related peptides such as relaxin that signal through G-protein-coupled receptors. After a half-century of advances in insulin chemistry, we have reached a point where synthesis is no longer limiting structural and biological investigation within this family of peptide hormones. The future will increasingly focus on the refinement of structure to meet medicinal purposes that have long been pursued, such as the development of a glucose-sensitive insulin. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.


Assuntos
Insulina/química , Peptídeos/síntese química , Relaxina/química , Somatomedinas/química , Animais , Dissulfetos/química , Humanos , Ligações de Hidrogênio , Estrutura Molecular , Dobramento de Proteína
7.
Artigo em Inglês | MEDLINE | ID: mdl-25899860

RESUMO

Insulin-like growth factor (Igf) is the key regulator for development, growth, and reproduction. In most vertebrate species, the Igf family has two forms: Igf1 and Igf2. A novel form of Igf, termed Igf3, was recently discovered in fish. In the present study, we isolated igf3 from the orange-spotted grouper (Epinephelus coioides). The orange-spotted grouper igf3 consisted of a full-length cDNA of 1014 nucleotides with an open reading frame (ORF) of 597 bp, encoding for proteins of 199 amino acid residues in length. Tissue distribution analysis showed that igf1 widely expressed with the highest expression in the pituitary and liver. igf2 was expressed highly in all the tissues except the olfactory bulb, while igf3 showed the highest expression in the ovary, and moderate expression in brain areas. The expression profiles of three igf genes during the ovarian development and growth hormone (Gh) and human chorionic gonadotropin (hCG) treatment were also investigated. Three igf genes exhibited different expression patterns during the ovarian development, and showed different responses to the Gh and hCG treatments, appearing to play distinct roles in ovarian development. The present study provides further evidence for the existence of an intraovarian Igf system in orange-spotted grouper.


Assuntos
Bass/genética , Bass/metabolismo , Proteínas de Peixes/genética , Somatomedinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bass/crescimento & desenvolvimento , Clonagem Molecular/métodos , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Somatomedinas/química , Somatomedinas/metabolismo
8.
Fish Physiol Biochem ; 41(3): 673-83, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25724869

RESUMO

A new cell line was established from half-smooth tongue sole Cynoglossus semilaevis pseudomale gonad (CSPMG). Primary culture was initiated from gonad tissues pieces, and the CSPMG cells were cultured at 24 °C in Dulbecco's modified Eagle medium/F12 medium (1:1) (pH7.0), supplemented with 20 % fetal bovine serum, basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-I, 2-mercaptoethanol, penicillin and streptomycin. The cultured CSPMG cells, in fibroblast shape, proliferated to 100 % confluency 10 days later and had been subcultured to passage 109. Chromosome analyses indicated that the CSPMG cells exhibited chromosomal aneuploidy with a modal chromosome number of 42, which displayed the normal diploid karyotype of half-smooth tongue sole (2n = 42t, NF = 42). Reverse transcription polymerase chain reaction revealed CSPMG cells could express gonad somatic cell functional genes Sox9a, Wt1a and weakly germ cell marker gene Vasa, but not male specific gene Dmrt1. Transfection experiment demonstrated that CSPMG cells transfected with pEGFP-N3 plasmid and small RNA could express green and red fluorescence signals with high transfection efficiency. In conclusion, a continuous CSPMG cell line has been established successfully. The cell line might serve as a valuable tool for studies on the mechanism of sex determination, sex reversal and gonad development in flatfish.


Assuntos
Técnicas de Cultura de Células/veterinária , Linhagem Celular , Meios de Cultura/química , Linguados , Gônadas/citologia , Animais , Técnicas de Cultura de Células/métodos , Proliferação de Células/fisiologia , Análise Citogenética/veterinária , Fator de Crescimento Epidérmico/química , Fator 2 de Crescimento de Fibroblastos/química , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/metabolismo , Mercaptoetanol/química , Penicilinas/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Fatores de Transcrição SOX9/metabolismo , Somatomedinas/química , Estreptomicina/química , Proteínas WT1/metabolismo
9.
Biomacromolecules ; 15(12): 4439-46, 2014 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-25329425

RESUMO

Sulfation patterns of glycosaminoglycans (GAG) govern the electrostatic complexation of biomolecules and thus allow for modulating the release profiles of growth factors from GAG-based hydrogels. To explore options related to this, selectively desulfated heparin derivatives were prepared, thoroughly characterized, and covalently converted with star-shaped poly(ethylene glycol) into binary polymer networks. The impact of the GAG sulfation pattern on the network characteristics of the obtained hydrogels was theoretically evaluated by mean field methods and experimentally analyzed by rheometry and swelling measurements. Sulfation-dependent differences of reactivity and miscibility of the heparin derivatives were shown to determine network formation. A theory-based design concept for customizing growth factor affinity and physical characteristics was introduced and validated by quantifying the release of fibroblast growth factor 2 from a set of biohybrid gels. The resulting new class of cell-instructive polymer matrices with tunable GAG sulfation will be instrumental for multiple applications in biotechnology and medicine.


Assuntos
Portadores de Fármacos/química , Fator 2 de Crescimento de Fibroblastos/química , Glicosaminoglicanos/química , Somatomedinas/química , Fenômenos Químicos , Heparina/química , Hidrogéis/química , Polietilenoglicóis/química , Espectrofotometria Infravermelho , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática
10.
J Anim Sci ; 92(4): 1800-7, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24663163

RESUMO

The 20 yr of commercial use of recombinant bovine somatotropin (rbST) in the United States provide the backdrop for reviewing the outcome of use on human health issues by the upcoming 78th meeting of the Joint Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) Expert Committee on Food Additives. These results and further advancements in scientific knowledge indicate there are no new human health issues related to the use of rbST by the dairy industry. Use of rbST has no effect on the micro- and macrocomposition of milk. Also, no evidence exists that rbST use has increased human exposure to antibiotic residues in milk. Concerns that IGF-I present in milk could have biological effects on humans have been allayed by studies showing that oral consumption of IGF-I by humans has little or no biological activity. Additionally, concentrations of IGF-I in digestive tract fluids of humans far exceed any IGF-I consumed when drinking milk. Furthermore, chronic supplementation of cows with rbST does not increase concentrations of milk IGF-I outside the range typically observed for effects of farm, parity, or stage of lactation. Use of rbST has not affected expression of retroviruses in cattle or posed an increased risk to human health from retroviruses in cattle. Furthermore, risk for development of type 1 or type 2 diabetes has not increased in children or adults consuming milk and dairy products from rbST-supplemented cows. Overall, milk and dairy products provide essential nutrients and related benefits in health maintenance and the prevention of chronic diseases.


Assuntos
Indústria de Laticínios , Hormônio do Crescimento/efeitos adversos , Hormônio do Crescimento/farmacologia , Leite/química , Animais , Antibacterianos/química , Bovinos , Criança , Resíduos de Drogas/análise , Feminino , Hormônio do Crescimento/administração & dosagem , Humanos , Recém-Nascido , Leite/citologia , Somatomedinas/química , Somatomedinas/metabolismo , Fatores de Tempo , Estados Unidos , Vírus
11.
Gen Comp Endocrinol ; 199: 56-64, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24503150

RESUMO

Hyperglycemia that is caused by the release of crustacean hyperglycemic hormone (CHH) from the sinus gland to hemolymph is one of the hallmark physiological phenomena, occurring in decapod crustaceans experiencing stressful conditions. However, the mechanism(s) by which such elevated glucose levels return to resting levels is still unknown. Interestingly, noted is a difference in the clearance rate of hemolymph glucose between adult females and adult males of the blue crab, Callinectes sapidus: the former with more rapid clearance than the latter. The presence of an endogenous-insulin-like molecule is suggested in C. sapidus because an injection of bovine insulin, significantly reduces the levels of hemolymph glucose that were previously elevated by emersion stress or the glucose injection. Using 5' and 3' RACE, the full-length cDNA of an insulin-like molecule is isolated from the hepatopancreas of an adult female C. sapidus and shows the same putative sequence of an insulin-like androgenic gland factor (IAG) but differs in 5' and 3' UTR sequences. A knock-down study using five injections of double-stranded RNA of CasIAG-hep (dsRNA-CasIAG-hep, 10µg/injection) over a 10-day period reduces CasIAG-hep expression by ∼50%. The levels of hemolymph glucose are also kept higher in dsRNA-CasIAG-hep injected group than those treated with dsRNA-green fluorescent protein (dsRNA-IAG-hep) or saline. Most importantly, the hepatopancreas of dsRNA-CasIAG-hep injected animals contains amounts of carbohydrate (glucose, trehalose, and glycogen) significantly lower than those of control groups, indicating that the function of CasIAG-hep in carbohydrate metabolism in crustaceans is similar to carbohydrate metabolism in vertebrates.


Assuntos
Braquiúros/metabolismo , Metabolismo dos Carboidratos , Hepatopâncreas/metabolismo , Somatomedinas/metabolismo , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes , Sequência de Bases , Braquiúros/efeitos dos fármacos , Braquiúros/genética , Metabolismo dos Carboidratos/efeitos dos fármacos , Metabolismo dos Carboidratos/genética , Bovinos , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Feminino , Perfilação da Expressão Gênica , Glucose/metabolismo , Hemolinfa/efeitos dos fármacos , Hemolinfa/metabolismo , Hepatopâncreas/efeitos dos fármacos , Imersão , Injeções , Insulina/administração & dosagem , Insulina/farmacologia , Hormônios de Invertebrado , Masculino , Dados de Sequência Molecular , Proteínas do Tecido Nervoso , RNA de Cadeia Dupla/metabolismo , Somatomedinas/química , Somatomedinas/genética
12.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi ; 31(6): 1319-24, 2014 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-25868252

RESUMO

Recombinant protein SMB(PRG4) containing two Somatomedin B domains and a small amount of glycosylation of repetitive sequences of proteoglycan 4 was cloned according to PGR4 gene polymorphism. Mature purification process was established and recombinant protein SMB(PRG4), with high-level expression was purified. By using size-exclusion chromatogaraphy and dynamic light scattering, we found that the recombinant protein self-aggregate to dimeric form. Structure prediction and non-reducing electrophoresis revealed that SMB(PRG4), was a non-covalently bonded dimer.


Assuntos
Proteoglicanas/química , Proteínas Recombinantes/química , Somatomedinas/química , Glicosilação , Multimerização Proteica
13.
Biochim Biophys Acta ; 1830(3): 2701-9, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23671931

RESUMO

BACKGROUND: Pregnancy-associated plasma protein-A (PAPP-A) is a local regulator of insulin-like growth factor (IGF) bioavailability in physiological systems, but many structural and functional aspects of the metzincin metalloproteinase remain to be elucidated. PAPP-A cleaves IGF binding protein (IGFBP)-4 and IGFBP-5. Cleavage of IGFBP-4, but not IGFBP-5, depends on the binding of IGF before proteolysis by PAPP-A can occur. The paralogue PAPP-A2 has two substrates among the six IGFBPs: IGFBP-3 and IGFBP-5. METHODS: Sets of chimeric proteins between IGFBP-4 and -5, and IGFBP-3 and -5 were constructed to investigate the structural requirements for IGF modulation. At the proteinase level, we investigated the importance of individual acidic amino acids positioned in the proteolytic domain of PAPP-A for proteolytic activity against IGFBP-4 and -5. Interaction between PAPP-A and its substrates was analyzed by surface plasmon resonance. RESULTS AND CONCLUSION: We provide data suggesting that the C-terminal domain of the IGFBPs is responsible for IGF-dependent modulation of access to the scissile bond. Loss or reduction of IGFBP proteolysis by PAPP-A was observed upon mutation of residues positioned in the unique 63-residue stretch separating the zinc and Met-turn motifs, and in the short sequence following the Met-turn methionine. A model of the proteolytic domain of PAPP-A suggests the presence of structural calcium ions in the C-terminal subdomain, implicated in IGFBP substrate interactions. GENERAL SIGNIFICANCE: Detailed knowledge of interactions between PAPP-A and its substrates is required to understand the modulatory role of PAPP-A on IGF receptor stimulation.


Assuntos
Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Proteína Plasmática A Associada à Gravidez/química , Somatomedinas/química , Sequência de Aminoácidos , Sítios de Ligação , Feminino , Células HEK293 , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Modelos Moleculares , Dados de Sequência Molecular , Gravidez , Proteína Plasmática A Associada à Gravidez/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Somatomedinas/genética , Especificidade por Substrato , Transfecção
14.
Mol Biol Rep ; 40(5): 3583-90, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23269623

RESUMO

Insulin-like growth factor peptides, play an important role in regulating cell growth, differentiation, and apoptosis, which has been demonstrated to promote the development of cancer. The purpose of our study is to assess the association between circulation insulin-like growth factor peptides and colorectal cancer (CRC) risk. We searched Medline, EMBASE, OVID and Web of Science and picked up epidemiological studies that satisfied our inclusion criteria. A meta-analysis of 19 epidemiological studies containing 5,155 cases and 9,420 controls related with the association of circulation insulin-like growth factor peptides and CRC risk was carried out. Meta-analysis showed that high level IGF-I and IGF-II significantly increased CRC risk, (OR = 1.25, 95% CI: 1.08-1.45 for IGF-I; OR = 1.52, 95% CI: 1.16-2.01 for IGF-II; OR = 0.85, 95% CI: 0.70-1.03 for IGFBP-1; OR = 0.77, 95% CI: 0.41-1.43 for IGFBP-2 and OR = 0.88, 95% CI: 0.71-1.10 for IGFBP-3). Subgroup analysis showed that the increased cancer risk by IGF-I was more distinguished in colon cancer (OR = 1.35, 95% CI: 1.04-1.75) and Caucasian (OR = 1.32, 95% CI: 1.12-1.56). Our meta-analysis provides comprehensive support for a role of circulation IGF-I and IGF-II in the etiology of CRC.


Assuntos
Neoplasias Colorretais/sangue , Neoplasias Colorretais/epidemiologia , Peptídeos/sangue , Risco , Somatomedinas/química , Humanos , Razão de Chances
15.
Biochemistry ; 51(41): 8256-66, 2012 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-22957734

RESUMO

The native fold of plasminogen activator inhibitor 1 (PAI-1) represents an active metastable conformation that spontaneously converts to an inactive latent form. Binding of the somatomedin B domain (SMB) of the endogenous cofactor vitronectin to PAI-1 delays the transition to the latent state and increases the thermal stability of the protein dramatically. We have used hydrogen/deuterium exchange mass spectrometry to assess the inherent structural flexibility of PAI-1 and to monitor the changes induced by SMB binding. Our data show that the PAI-1 core consisting of ß-sheet B is rather protected against exchange with the solvent, while the remainder of the molecule is more dynamic. SMB binding causes a pronounced and widespread stabilization of PAI-1 that is not confined to the binding interface with SMB. We further explored the local structural flexibility in a mutationally stabilized PAI-1 variant (14-1B) as well as the effect of stabilizing antibody Mab-1 on wild-type PAI-1. The three modes of stabilizing PAI-1 (SMB, Mab-1, and the mutations in 14-1B) all cause a delayed latency transition, and this effect was accompanied by unique signatures on the flexibility of PAI-1. Reduced flexibility in the region around helices B, C, and I was seen in all three cases, which suggests an involvement of this region in mediating structural flexibility necessary for the latency transition. These data therefore add considerable depth to our current understanding of the local structural flexibility in PAI-1 and provide novel indications of regions that may affect the functional stability of PAI-1.


Assuntos
Espectrometria de Massas/métodos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Somatomedinas/metabolismo , Vitronectina/metabolismo , Deutério , Hidrogênio , Modelos Moleculares , Inibidor 1 de Ativador de Plasminogênio/química , Somatomedinas/química , Vitronectina/química
16.
J Am Chem Soc ; 134(15): 6579-83, 2012 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-22471692

RESUMO

Adsorption of biomolecules at metal surfaces often creates two-dimensional ordering of the adlayers. However, metal substrate reconstruction is less commonly observed, unless upon annealing of the molecule-surface system. Here, we report on the drastic room-temperature reconstruction of the Au(111) surface, driven by the adsorption of insulin growth factor tripeptide molecules. Scanning tunneling microscopy images show that the surface reconstruction, which takes place without annealing the system, is dynamic and evolves over time. It is initiated at kinks and steps edges, but the reconstruction also takes place within defect-free terraces. Theoretical calculations are performed to explain the reconstruction at the molecular level.


Assuntos
Ouro/química , Somatomedinas/química , Adsorção , Oligopeptídeos/química , Propriedades de Superfície
17.
J Cell Physiol ; 227(11): 3566-74, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22262087

RESUMO

The homodimerization of ENPP1 is mediated by the two somatomedin B (SMB) domains of the protein through a mechanism that is yet unknown at the atomistic level. The tandem arrangement of these domains without an intermediate spacer implies their possible packing into a functional assembly, which we explored by rigid docking. To exclude potential bias in the docking search we assessed the absence of flexible protein regions by evaluating the normalized B-factors calculated from the Cα atom displacements derived from molecular dynamics simulations. After filtering the docking results exploiting the criterion that residues located at the inter-domain interfaces are more conserved than non-interface residues, the resulting best model of the tandem SMB domains revealed the presence of two large conserved surface patches not engaged in the inter-domain contact. The largest patch is flat and contains all the invariant positively charged residues characterized by fully solvent-exposed side chains within the tandem SMB domains, suggesting as a possible role its interaction with the negative phospholipids on the cell surface. We envisage that an ENPP1 monomer bound to the cell membrane via the transmembrane segment can also interact with the cell surface through the largest conserved patch favoring a specific geometry of the tandem SMB module on the cell that optimally exposes the second conserved patch for the symmetric interaction with another membrane-bound ENPP1 monomer, finally promoting the homodimerization. Biological implications of this model and insights into the effects of the K173Q variant associated with insulin resistance and related abnormalities are presented.


Assuntos
Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Domínios e Motivos de Interação entre Proteínas , Pirofosfatases/química , Pirofosfatases/metabolismo , Somatomedinas , Sequência de Aminoácidos , Animais , Carbono/química , Variação Genética , Humanos , Resistência à Insulina/genética , Resistência à Insulina/fisiologia , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Diester Fosfórico Hidrolases/genética , Conformação Proteica , Multimerização Proteica , Pirofosfatases/genética , Alinhamento de Sequência , Somatomedinas/química , Somatomedinas/genética , Somatomedinas/metabolismo
18.
J Biol Chem ; 286(50): 43515-26, 2011 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-22025616

RESUMO

The high-affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays a regulatory role for both extravascular fibrinolysis and uPAR-mediated adhesion and migration on vitronectin-coated surfaces. We have recently proposed that the adhesive function of uPAR is allosterically regulated via a "tightening" of its three-domain structure elicited by uPA binding. To challenge this proposition, we redesigned the uPAR structure to limit its inherent conformational flexibility by covalently tethering domains DI and DIII via a non-natural interdomain disulfide bond (uPAR(H47C-N259C)). The corresponding soluble receptor has 1) a smaller hydrodynamic volume, 2) a higher content of secondary structure, and 3) unaltered binding kinetics towards uPA. Most importantly, the purified uPAR(H47C-N259C) also displays a gain in affinity for the somatomedin B domain of vitronectin compared with uPAR(wt), thus recapitulating the improved affinity that accompanies uPA-uPAR(wt) complex formation. This functional mimicry is, intriguingly, operational also in a cellular setting, where it controls lamellipodia formation in uPAR-transfected HEK293 cells adhering to vitronectin. In this respect, the engineered constraint in uPAR(H47C-N259C) thus bypasses the regulatory role of uPA binding, resulting in a constitutively active uPAR. In conclusion, our data argue for a biological relevance of the interdomain dynamics of the glycolipid-anchored uPAR on the cell surface.


Assuntos
Pseudópodes/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Linhagem Celular , Cromatografia em Gel , Dicroísmo Circular , Drosophila , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Somatomedinas/química , Somatomedinas/metabolismo , Ressonância de Plasmônio de Superfície , Vitronectina/química , Vitronectina/metabolismo
19.
Curr Drug Targets ; 12(12): 1729-43, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21707478

RESUMO

Urokinase-type plasminogen activator (uPA) is one of the two physiological serine proteases responsible for the activation of plasminongen to plasmin. uPA activity is regulated by its inhibitors (PAI-1 and PAI-2) and its receptor (uPAR), and an expanding list of their interacting proteins. In addition to plasminogen activation, this system also plays important roles in the regulation of many cellular processes including cell proliferation, adhesion and migration. It is beyond reasonable doubt that this enzyme system plays a central role in tumor biology and represents a high potential target for therapeutic intervention of tumor growth and metastasis. During the past fifteen years, crystal structures of uPA and its inhibitors have facilitated the development of uPA inhibitors. Many crystal structures of proteins in the uPA/uPAR system have also been reported recently, especially a series of structures of uPAR and its complexes with vitronectin and uPA, facilitating the development and evaluation of uPAR inhibitors. Recent progress on uPA inhibitors will be summarized in this article. The unique structural features and the druggable potentials of these new structures will also be discussed.


Assuntos
Terapia de Alvo Molecular , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Disponibilidade Biológica , Humanos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacocinética , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/uso terapêutico , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Transdução de Sinais/efeitos dos fármacos , Somatomedinas/química , Somatomedinas/metabolismo , Somatomedinas/farmacologia , Somatomedinas/uso terapêutico , Pesquisa Médica Translacional , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
20.
Protein Sci ; 20(2): 353-65, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21280127

RESUMO

Human plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor with a metastable active conformation. Under physiological conditions, half of the inhibitor transitions to a latent state within 1-2 h. The interaction between PAI-1 and the plasma protein vitronectin prolongs this active lifespan by ∼50%. Previously, our group demonstrated that PAI-1 binds to resins using immobilized metal affinity chromatography (Day, U.S. Pat. 7,015,021 B2, March 21, 2006). In this study, the effect of these metals on function and stability was investigated by measuring the rate of the transition from the active to latent conformation. All metals tested showed effects on stability, with the majority falling into one of two types depending on their effects. The first type of metal, which includes magnesium, calcium and manganese, invoked a slight stabilization of the active conformation of PAI-1. A second category of metals, including cobalt, nickel and copper, showed the opposite effects and a unique vitronectin-dependent modulation of PAI-1 stability. This second group of metals significantly destabilized PAI-1, although the addition of vitronectin in conjunction with these metals resulted in a marked stabilization and slower conversion to the latent conformation. In the presence of copper and vitronectin, the half-life of active PAI-1 was extended to 3 h, compared to a half-life of only ∼30 min with copper alone. Nickel had the largest effect, reducing the half-life to ∼5 min. Together, these data demonstrate a heretofore-unknown role for metals in modulating PAI-1 stability.


Assuntos
Cálcio/metabolismo , Magnésio/metabolismo , Metais Pesados/metabolismo , Inibidor 1 de Ativador de Plasminogênio/química , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Sítios de Ligação , Cálcio/química , Cloretos/química , Cloretos/metabolismo , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Humanos , Cinética , Magnésio/química , Metais Pesados/química , Estabilidade Proteica , Somatomedinas/química , Somatomedinas/metabolismo , Vitronectina/química , Vitronectina/metabolismo
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