RESUMO
Transferrin binding protein A (TbpA) is a TonB-dependent outer membrane protein expressed by pathogenic bacteria for iron acquisition from human transferrin. The N-terminal 160 residues (plug domain) of TbpA were overexpressed in both the periplasm and cytoplasm of Escherichia coli. We found this domain to be soluble and monodisperse in solution, exhibiting secondary structure elements found in plug domains of structurally characterized TonB-dependent transporters. Although the TbpA plug domain is apparently correctly folded, we were not able to observe an interaction with human transferrin by isothermal titration calorimetry or nitrocellulose binding assays. These experiments suggest that the plug domain may fold independently of the beta-barrel, but extracellular loops of the beta-barrel are required for ligand binding.
Assuntos
Neisseria meningitidis/química , Estrutura Secundária de Proteína , Proteína A de Ligação a Transferrina/química , Animais , Dicroísmo Circular , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Mutação , Estrutura Terciária de Proteína , Proteína A de Ligação a Transferrina/genéticaRESUMO
This study reports the tissue distribution of Hepatocyte Growth Factor-Scatter Factor (HGF/SF) in human fetal tissue using Northern analysis and in situ hybridisation techniques. In tissue from fetuses of 9-17 wk gestational age, the 6 kb mRNA transcript for HGF/SF was demonstrated in many tissues but prominently in liver, intestine, gall bladder and spleen. In situ hybridisation demonstrated that HGF/SF expression was not confined to mesenchymal tissues, as suggested by previous studies but was expressed in epithelial tissues, particularly in small intestine, keratinising epithelium of tongue, skin and oesophagus. In the small intestine epithelial expression was strikingly regional, being confined to the crypt region, the site of enterocyte proliferation. Northern analysis of tissues for c-met mRNA, representing expression of the HGF/SF receptor, demonstrated receptor expression in all tissues studied except the thyroid gland.
Assuntos
Northern Blotting , Desenvolvimento Embrionário e Fetal/fisiologia , Fator de Crescimento de Hepatócito/metabolismo , Hibridização In Situ , Mesoderma/metabolismo , Epitélio/química , Epitélio/embriologia , Epitélio/metabolismo , Esôfago/química , Esôfago/embriologia , Vesícula Biliar/química , Vesícula Biliar/embriologia , Fator de Crescimento de Hepatócito/análise , Humanos , Intestinos/química , Intestinos/embriologia , Fígado/química , Fígado/embriologia , Mesoderma/química , Proteínas Proto-Oncogênicas c-met , Receptores Proteína Tirosina Quinases/metabolismo , Pele/química , Pele/embriologia , Baço/química , Baço/embriologia , Língua/química , Língua/embriologiaRESUMO
We have quantified mRNA for the hepatocyte growth factor and its putative receptor the c-met proto-oncogene protein product, in a series of human primary and secondary liver tumours and adjacent non-neoplastic liver. In all hepatocellular cancers, hepatocyte growth factor 6 kb mRNA expression was less (mean 23.93% +/- 6.33% S.E.M. n = 7) in the tumours than in the adjacent normal liver. Both relative over- and under-expression of c-met transcripts were found in tumour tissue compared to non-neoplastic liver. Thus hepatocellular cancer tissue does not over-express mRNA for hepatocyte growth factor, though this growth factor might play a role in hyperproliferative states leading to liver cancer.
Assuntos
Adenoma/química , Carcinoma Hepatocelular/química , Fator de Crescimento de Hepatócito/genética , Neoplasias Hepáticas/química , RNA Mensageiro/análise , Receptores Proteína Tirosina Quinases/genética , Adenoma/genética , Sequência de Bases , Northern Blotting , Carcinoma Hepatocelular/genética , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Fator de Crescimento de Hepatócito/análise , Humanos , Fígado/química , Neoplasias Hepáticas/genética , Dados de Sequência Molecular , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-met , RNA Mensageiro/genética , Receptores Proteína Tirosina Quinases/análise , Transcrição GênicaRESUMO
Wild-type and site-specific mutants C166S and C166A (Cys-166-->Ser and Cys-166-->Ala respectively) of the amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were expressed in Escherichia coli by using the vector pKK223-3. Both mutant proteins were catalytically inactive but showed complete cross-reactivity with polyclonal antiserum raised against the wild-type enzyme, as well as CD spectra identical with that of the wild-type enzyme, which were indicative of correct folding. Cys-166 is therefore implicated as the active-site nucleophile. Titration of free thiol groups with 5,5'-dithiobis-(2-nitrobenzoic acid) indicated that Cys-166 is not a rapidly reacting residue. Crystals of both wild-type and C166S amidase grew with identical, rhombohedral morphology; X-ray diffraction analysis established the unit cell dimensions (a=b=c=84 A; alpha=beta=gamma=75 degrees) and space group (R3 or R32). These results imply a quaternary structure of six subunits, with most probably 32 symmetry; the existence of a hexameric structure was supported by molecular mass determinations based on gel filtration and electrophoretic mobility.