RESUMO
G protein-coupled receptors (GPCRs) are membrane proteins that mediate signaling across the cellular membrane and facilitate cellular responses to external stimuli. Due to the critical role that GPCRs play in signal transduction, therapeutics have been developed to influence GPCR function without an extensive understanding of the receptors themselves. Closing this knowledge gap is of paramount importance to improving therapeutic efficacy and specificity, where efforts to achieve this end have focused chiefly on improving our knowledge of the structure-function relationship. The purpose of this chapter is to review methods for the heterologous expression of GPCRs in Saccharomyces cerevisiae, including whole-cell assays that enable quantitation of expression, localization, and function in vivo. In addition, we describe methods for the micellular solubilization of the human adenosine A2a receptor and for reconstitution of the receptor in liposomes that have enabled its biophysical characterization.
Assuntos
Receptores Acoplados a Proteínas G/genética , Saccharomyces cerevisiae/genética , Animais , Vetores Genéticos/genética , Humanos , Plasmídeos/genética , Receptores Acoplados a Proteínas G/análise , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The advancement of nonviral gene therapy hinges on the ability to exert highly specific spatial and temporal control of gene delivery systems to enable localized release of DNA. In this work, we have developed a system capable of promoting localized delivery of a plasmid by utilizing peptide nucleic acid (PNA) technology to bind DNA to a substrate via an enzymatically labile peptide sequence. The successful immobilization of the DNA to the model substrate as well as the specificity of the binding was confirmed with atomic force microscopy (AFM) and AFM-confocal overlay imaging. Fluorescence-based quantification revealed that surfaces treated with the conjugates had 49 ± 22 ng of DNA/cm(2), while there were 4.2 ± 2.1 ng of DNA/cm(2) on surfaces treated with unfunctionalized DNA. When NIH/3T3 cells were grown on the modified substrates, a significantly higher percentage of cells were transfected when the peptide tether was protease-sensitive as compared with when it was not labile. These results indicated that the peptide must be cleaved to release the DNA. In addition to providing cell-triggered release, this system decouples the properties of the complexation agent and the substrate from the method of immobilization/release to provide a model system that can be tailored to specific applications.
Assuntos
DNA Viral/química , DNA Viral/genética , Técnicas de Transferência de Genes , Ácidos Nucleicos Peptídicos/química , Plasmídeos/química , Plasmídeos/genética , Animais , Células Cultivadas , Camundongos , Células NIH 3T3 , Propriedades de SuperfícieRESUMO
Polycationic polymers have been used to condense therapeutic DNA into submicron particles, offering protection from shear-induced or enzymatic degradation. However, the spontaneous nature of this self-assembly process gives rise to the formation of multimolecular aggregates, resulting in significant polyplex heterogeneity. Additionally, cytotoxicity issues and serum instability have limited the in vivo efficacy of such systems. One way these issues can be addressed is through the inclusion of poly(ethylene glycol) (PEG). PEG has known steric effects that inhibit polyplex self-aggregation. A variety of PEGylated gene delivery formulations have been previously pursued in an effort to take advantage of this material's benefits. Because of such interest, our aim was to further explore the consequences of PEG inclusion on the structure and activity of gene delivery vehicle formulations. We explored the complexation of plasmid DNA with varying ratios of a PEGylated trilysine peptide (PEG-K(3)) and 25-kDa polyethylenimine (PEI). Atomic force and scanning electron microscopy were utilized to assess the polyplex size and shape and revealed that a critical threshold of PEG was necessary to promote the formation of homogeneous polyplexes. Flow cytometry and fluorescence microscopy analyses suggested that the presence of PEG inhibited transfection efficiency as a consequence of changes in intracellular trafficking and promoted an increased reliance on energy-independent mechanisms of cellular uptake. These studies provide new information on the role of PEG in delivery vehicle design and lay the foundation for future work aimed at elucidating the details of the intracellular transport of PEGylated polyplexes.