RESUMO
Melanocytes develop from the vertebrate embryo-specific neural crest, migrate, and localize in various organs, including not only the skin but also several extracutaneous locations such as the heart, inner ear and choroid. Little is known about the functions of extracutaneous melanocytes except for cochlear melanocytes, which are essential for hearing ability. In this study, we focused on the structure of the choroid, in which melanocytes are abundant around the well-developed blood vascular system. By comparing structural differences in the choroid of wild-type and melanocyte-deficient Mitfmi-bw/Mitfmi-bw mutant mice, our observations suggest that choroidal melanocytes contribute to the morphogenesis and/or maintenance of the normal vasculature structure of that tissue.
Assuntos
Corioide/fisiologia , Melanócitos/fisiologia , Animais , Corioide/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Neovascularização Fisiológica/fisiologiaRESUMO
Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that reside in the target membranes and transport vesicles assemble into specific SNARE complexes to drive membrane fusion. N-ethylmaleimide-sensitive factor (NSF) and its attachment protein, α-SNAP (encoded by NAPA), catalyze disassembly of the SNARE complexes in the secretory and endocytic pathways to recycle them for the next round of fusion events. γ-SNAP (encoded by NAPG) is a SNAP isoform, but its function in SNARE-mediated membrane trafficking remains unknown. Here, we show that γ-SNAP regulates the endosomal trafficking of epidermal growth factor (EGF) receptor (EGFR) and transferrin. Immunoprecipitation and mass spectrometry analyses revealed that γ-SNAP interacts with a limited range of SNAREs, including endosomal ones. γ-SNAP, as well as α-SNAP, mediated the disassembly of endosomal syntaxin-7-containing SNARE complexes. Overexpression and small interfering (si)RNA-mediated depletion of γ-SNAP changed the morphologies and intracellular distributions of endosomes. Moreover, the depletion partially suppressed the exit of EGFR and transferrin from EEA1-positive early endosomes to delay their degradation and uptake. Taken together, our findings suggest that γ-SNAP is a unique SNAP that functions in a limited range of organelles - including endosomes - and their trafficking pathways.
Assuntos
Endocitose/fisiologia , Receptores ErbB/metabolismo , Transporte Proteico/fisiologia , Proteínas Qa-SNARE/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/genética , Membrana Celular/metabolismo , Endossomos/metabolismo , Células Hep G2 , Humanos , Fusão de Membrana/fisiologia , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno , Transferrina/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
We have done conformational study of hNck2 SH3 domain by means of far-ultraviolet (far-UV) circular dichroism (CD) and X-ray solution scattering (XSS). The results indicated that the following: (1) hNck2 SH3 domain protein exhibited concentration dependent monomer-dimer transition at neutral pH, while the secondary structure of this protein was independent of the protein concentration. (2) The hNck2 SH3 domain also exhibited pH dependent monomer-dimer transition. This monomer-dimer transition was accompanied with helix-ß transition of the secondary structural change. Moreover, the acid-induced conformation, which was previously studied by Liu and Song by CD and nuclear magnetic resonance (NMR), was found to be not compact, but the conformation of the protein at acidic pH was similar to the cold denatured state (C-state) reported by Yamada et al. for equine ß-lactoglobulin. We calculated that a structure of the equilibrium helix-rich intermediate of the hNck2 SH3 domain by DAMMIF program.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Oncogênicas/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Dicroísmo Circular , Dimerização , Humanos , Concentração de Íons de Hidrogênio , Proteínas Oncogênicas/metabolismo , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios X , Domínios de Homologia de srcRESUMO
Human transferrin (Tf) very tightly binds two ferric ions to deliver iron to cells. Fe(III)(2)Tf (Fe(2)Tf) binds to the Tf receptor (TfR) at pH 7.4; however, iron-free Tf (apoTf) does not. Iron uptake is facilitated by endocytosis of the Fe(2)Tf-TfR complex. Tf can also bind aluminum ions, which cause toxic effects and are associated with many diseases. Since Al(III)(2)Tf (Al(2)Tf) does not bind to TfR, the uptake of aluminum by the cells does not occur through a TfR-mediated pathway. We have studied the absence of binding between Al(2)Tf and TfR by investigating the physicochemical characteristics of apoTf, Al(2)Tf, Fe(2)Tf, and TfR. The hydrodynamic radius of 38.8 A for Al(2)Tf obtained by dynamic light scattering was between that of 42.6 A for apoTf and 37.2 A for Fe(2)Tf. The zeta potential of -11.3 mV for Al(2)Tf measured by capillary electrophoresis was close to -11.2 mV for apoTf as compared to -11.9 mV for Fe(2)Tf, indicating that the Al(2)Tf surface had a relatively scarce negative charge as the apoTf surface had. These results demonstrated that the structure of Al(2)Tf was a trade-off between the closed and open forms of Fe(2)Tf and apoTf, respectively. Consequently, it is suggested that Al(2)Tf cannot form specific ionic interresidual interactions, such as those formed by Fe(2)Tf, to bind to TfR, resulting in impossible complex formation between Al(2)Tf and TfR.
Assuntos
Alumínio/química , Receptores da Transferrina/química , Transferrina/química , Alumínio/metabolismo , Sítios de Ligação , Ligação Competitiva , Compostos Férricos/química , Compostos Férricos/metabolismo , Compostos Ferrosos/química , Compostos Ferrosos/metabolismo , Humanos , Ferro/química , Ferro/metabolismo , Cinética , Compostos Organometálicos/química , Compostos Organometálicos/metabolismo , Ligação Proteica , Receptores da Transferrina/metabolismo , Espectrofotometria , Oligoelementos/química , Oligoelementos/metabolismo , Transferrina/metabolismoRESUMO
Active Hexose Correlated Compound (AHCC), an extract derived from fungi of Basidiomycetes family has been shown to act as a biological response modifier in various disorders. In our present study, ferric nitrilotriacetate (Fe-NTA), which generates hydroxyl radicals in vivo, was given intraperitoneally to rats and AHCC was tested for its ability to suppress oxidative stress and the activity of ornithine decarboxylase (ODC) in the liver and kidney. Substantial increments in glutathione-related enzymes including glutathione reductase, glutathione peroxidase activity as well as oxidized glutathione contents were shown in the liver at 12 h after treatment with Fe-NTA (7.5 mg Fe/kg body weight). Effects of oxidative stress induced by Fe-NTA were also demonstrated by the increase in serum lipid peroxidation, aminotransferases and urinary 8-hydroxy-2'-deoxyguanosine. However, the increases in these parameters were restored to normal in AHCC-pretreated rats. The ODC activity in the liver and kidney was significantly increased by Fe-NTA, while the increased ODC activity induced by Fe-NTA was normalized in AHCC-pretreated rats. These results suggest AHCC acts as a potent antioxidant and protects against disorders induced by oxidative stresses.