RESUMO
Conformations of the prototypic UCP-1 (uncoupling protein-1) and its TM (transmembrane) and ML (matrix-loop) domains were studied by CD spectroscopy. Recombinant, untagged mouse UCP-1 and a hexahistidine-tagged version of the protein were obtained in high purity following their overexpression in Escherichia coli. The TM and ML domains of hamster UCP-1 were chemically synthesized. Conformations of both recombinant UCP-1 proteins were dominantly helical (40-50%) in digitonin micelles. Binding of the purine nucleotides GDP and GTP to UCP-1, detected in the near-UV CD region, supported the existence of the functional form of the protein in digitonin micelles. All individual TM and ML peptides, except the third ML domain, adopted helical structures in aqueous trifluoroethanol, which implies that, in addition to six TM segments, at least two of the ML domains of the UCP-1 can form helical structures in membrane interface regions. TM and ML domains interacted with vesicles composed of the main phospholipids of the inner membrane of mitochondria, phosphatidylcholine, phosphatidylethanolamine and cardiolipin, to adopt dominantly beta- and/or unordered conformations. Mixtures of UCP-1 peptide domains spontaneously associated in aqueous, phospholipid vesicles and digitonin micelle environments to form ordered conformations, which exhibited common features with the conformations of the full-length proteins. Thermal denaturations of UCP-1 and its nine-peptide-domain assembly in digitonin were co-operative but not reversible. Assembly of six TM domains in lipid bilayers formed ion-conducting units with possible helical bundle conformations. Consequently, covalent connection between peptide domains, tight domain interactions and TM potential are essential for the formation of the functional conformation of UCP-1.
Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Cricetinae , Eletrofisiologia , Expressão Gênica , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Canais Iônicos/genética , Canais Iônicos/isolamento & purificação , Mesocricetus , Camundongos , Micelas , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/isolamento & purificação , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Temperatura , Proteína Desacopladora 1RESUMO
BACKGROUND: Chemokines are small proteins that signal to and attract cells of the immune system; they are vital components in the modulation of immunity and wound healing. A newly described chemokine was reported to have antibacterial and antifungal activity. This chemokine, chemokine (C-C motif) ligand 28 (CCL28; also called mucosae-associated epithelial chemokine), is secreted by mucosal epithelial cells and is found in saliva and in breast milk. The objective of this study was to test whether CCL28 has antibacterial activity against two anaerobic periodontal pathogens: Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans. METHODS: We used a bacterial viability test, in which two fluorescent dyes are bound differentially to living and killed bacteria. We tested the bacteria at concentrations of 2 x 10(7)/ml, exposing them to CCL28 at concentrations from 0.04 to 10 microM. RESULTS: CCL28 was effective at killing both organisms. After 1 hour of exposure to the chemokine under appropriate oxygen conditions, the percentage of living organisms was reduced significantly for each species. We estimated the 50% effective concentration to be approximately 0.7 microM for P. gingivalis and approximately 2.0 microM for A. actinomycetemcomitans (N = five experiments each). We confirmed these observations using standard bacterial plating methods. CONCLUSION: Because this chemokine is secreted into the saliva, a reduction in salivary flow (as in xerostomia) may diminish the oral self-defense mechanisms by also reducing the exposure of bacteria to the antibacterial action of CCL28.
Assuntos
Aggregatibacter actinomycetemcomitans/efeitos dos fármacos , Anti-Infecciosos Locais/farmacologia , Quimiocinas CC/farmacologia , Porphyromonas gingivalis/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Contagem de Colônia Microbiana , Humanos , Testes de Sensibilidade Microbiana , Nefelometria e Turbidimetria , Proteínas Recombinantes/farmacologia , Saliva/químicaAssuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Bactérias Gram-Negativas/metabolismo , Fímbrias Bacterianas/metabolismo , Bactérias Gram-Negativas/patogenicidade , Proteínas de Membrana Transportadoras/metabolismo , Transporte Proteico , Virulência , Fatores de Virulência/metabolismoRESUMO
A number of protein secretion mechanisms have been identified in gram-negative pathogens. Many of these secretion systems are dependent upon the Sec translocase for protein export from the cytoplasm into the periplasm and then utilize other mechanisms for transport from the periplasm through the outer membrane. In this article, we review secretion similarities between autotransporter and two-partner secretion systems, and we report similarities between the autotransporter secretion mechanism with that of intimin/invasins. Considering that many secreted proteins are virulence factors, a better understanding of their secretion mechanisms will aid in the development of disease treatments and new bacterial vaccines.
