RESUMO
The study of dynamic movement and interactions of proteins inside living cells in real time is critical for a better understanding of cellular mechanisms and functions in molecular detail. Genetically encoded fusions to fluorescent protein(s) (FP) have been widely used for this purpose [Annu. Rev. Biochem. 1998, 67, 509-544]. To obviate some of the drawbacks associated with the use of FPs [Curr. Opin. Biotechnol. 2005, 16, 1-6; Nat. Methods2006, 3, 591-596], we report a small molecule-based approach that exploits the unique reactivity between the cysteine residue at the N-terminus of a target protein and cell-permeable, thioester-based small molecule probes resulting in site-specific, covalent tagging of proteins. This approach has been demonstrated by the in vivo labeling of proteins in both bacterial and mammalian systems thereby making it potentially useful for future bioimaging applications.
Assuntos
Sondas Moleculares/química , Proteínas Recombinantes de Fusão/análise , Linhagem Celular , Cisteína/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Humanos , Inteínas , Microscopia de Fluorescência , Sondas Moleculares/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
In the advent of an influenza virus pandemic it is likely that the administration of antiviral drugs will be an important first line of defence against the virus. The drugs currently in use are effective against seasonal influenza virus infection, and some cases have been used in the treatment of patients infected with the avian H5N1 influenza virus. However, it is becoming clear that the emergence of drug-resistant viruses will potentially be a major problem in the future efforts to control influenza virus infection. In addition, during a new pandemic, sufficient quantities of these agents will need to be distributed to many different parts of the world, possibly at short notice. In this review we provide an overview of some of the drugs that are currently available for the treatment and prevention of influenza virus infection. In addition, basic research on influenza virus is providing a much better understanding of the biology of the virus, which is offering the possibility of new anti-influenza virus drugs. We therefore also review some new antiviral strategies that are being reported in the scientific literature, which may form the basis of the next generation of antiviral strategies during a future influenza virus pandemic.
Assuntos
Antivirais/uso terapêutico , Surtos de Doenças/estatística & dados numéricos , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/prevenção & controle , Influenza Humana/tratamento farmacológico , Amantadina/uso terapêutico , Animais , Aves , Saúde Global , Humanos , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Oseltamivir/uso terapêutico , RNA Interferente PequenoRESUMO
One of the critical issues in the generation of a protein microarray lies in the choice of immobilization strategies, which ensure proteins are adhered to the glass surface while properly retaining their native biological activities. Herein, we report a bacterium-based, intein-mediated strategy to generate N-terminal cysteine-containing proteins which are then chemoselectively immobilized to a thioester-functionalized glass slide to generate the corresponding protein microarray. We also showed preliminary data of the strategy in a yeast host system.
Assuntos
Inteínas , Análise Serial de Proteínas , Proteínas/química , Eletroforese em Gel de PoliacrilamidaRESUMO
The post-genomic era heralds a multitude of challenges for chemists and biologists alike, with the study of protein functions at the heart of much research. The elucidation of protein structure, localization, stability, post-translational modifications, and protein interactions will steadily unveil the role of each protein and its associated biological function in the cell. The push to develop new technologies has necessitated the integration of various disciplines in science. Consequently, the role of chemistry has never been so profound in the study of biological processes. By combining the strengths of recombinant DNA technology, protein splicing, organic chemistry, and the chemoselective chemistry of native chemical ligation, various strategies have been successfully developed and applied to chemoselectively label proteins, both in vitro and in live cells, with biotin, fluorescent, and other small molecule probes. The site-specific incorporation of molecular entities with unique chemical functionalities in proteins has many potential applications in chemical and biological studies of proteins. In this article, we highlight recent progress of these strategies in several areas related to proteomics and chemical biology, namely, in vitro and in vivo protein biotinylation, protein microarray technologies for large-scale protein analysis, and live-cell bioimaging.
Assuntos
Bioquímica , Proteínas/metabolismo , Proteômica , Fenômenos Bioquímicos , Biotina/metabolismo , Inteínas/fisiologia , Ligantes , Análise Serial de ProteínasRESUMO
We review various advancements in small molecule probes, intein-based labeling methods, and the incorporation of synthetic amino acids into proteins for live cell imaging experiments. Finally, recent developments in quantum dots-based labeling are briefly reviewed.
Assuntos
Células/citologia , Diagnóstico por Imagem , Animais , Técnicas Citológicas , Corantes Fluorescentes , Humanos , Sondas Moleculares/química , NanotecnologiaRESUMO
Recent advances in the generation of peptide and protein microarrays are reviewed, with special focuses on different strategies available for site-specific immobilization of proteins and peptides.
Assuntos
Análise Serial de Proteínas/métodos , Avidina , Biotinilação , DNA Polimerase Dirigida por DNA/química , Peptídeos/química , Proteínas/química , Proteômica , PuromicinaRESUMO
We have successfully synthesized a number of small molecule probes designed for site-specific labeling of N-terminal cysteine-containing proteins expressed in live cells. Their utility for site-specific, covalent modifications of proteins was successfully demonstrated with purified proteins in vitro, and with live bacterial cells in vivo.