RESUMO
Viperin (virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible) is a widely distributed protein that is expressed in response to infection and causes antiviral effects against a broad spectrum of viruses. Viperin is a member of the radical S-adenosyl-l-methionine (SAM) superfamily of enzymes, which typically employ a 4Fe-4S cluster to reductively cleave SAM to initiate chemistry. Though the specific reaction catalyzed by viperin remains unknown, it has been shown that expression of viperin causes an increase in the fluidity of lipid membranes, which impedes the budding of nascent viral particles from the membrane inhibiting propagation of the infection. Herein, we show that expression of the human viperin homologue induces a dramatically elongated morphology of the host Escherichia coli cells. Mutation of an essential cysteine that coordinates the radical SAM cluster abrogates this effect. Thus, the native radical SAM activity of viperin is likely occurring in the host bacteria, indicating the elusive substrate is shared between both bacteria and humans, significantly narrowing the range of potential candidate substrates and providing a convenient bacterial platform from which future studies can occur.
Assuntos
Escherichia coli/fisiologia , Proteínas/fisiologia , S-Adenosilmetionina/metabolismo , Substituição de Aminoácidos , Aderência Bacteriana , Cisteína/química , Escherichia coli/citologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Cinética , Microscopia Confocal , Mutagênese Sítio-Dirigida , Mutação , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/genética , Proteínas Periplásmicas de Ligação/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteínas/química , Proteínas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Understanding the dynamics of chemoattractant signaling is key to our understanding of the mechanisms underlying the directed migration of cells, including that of neutrophils to sites of infections and of cancer cells during metastasis. A model frequently used for deciphering chemoattractant signal transduction is the social amoeba Dictyostelium discoideum. However, the methods available to quantitatively measure chemotactic signaling are limited. Here, we describe a protocol to quantitatively study chemoattractant signal transduction in Dictyostelium by monitoring protein-protein interactions and conformational changes using Bioluminescence Resonance Energy Transfer (BRET).