Split Luciferase as an Optical Probe for Detecting Protein−Protein Interactions in Mammalian Cells Based on Protein Splicing

Dengue virus (DENV), an emerging mosquito-transmitted pathogen capable of causing severe disease in humans, interacts with host cell factors to create a more favorable environment for replication. However, few interactions between DENV and human proteins have been reported to date. To identify DENV-human protein interactions, we used high-throughput yeast two-hybrid assays to screen the 10 DENV proteins against a human liver activation domain library. From 45 DNA-binding domain clones containing either full-length viral genes or partially overlapping gene fragments, we identified 139 interactions between DENV and human proteins, the vast majority of which are novel. These interactions involved 105 human proteins, including six previously implicated in DENV infection and 45 linked to the replication of other viruses. Human proteins with functions related to the complement and coagulation cascade, the centrosome, and the cytoskeleton were enriched among the DENV interaction partners. To determine if the cellular proteins were required for DENV infection, we used small interfering RNAs to inhibit their expression. Six of 12 proteins targeted (CALR, DDX3X, ERC1, GOLGA2, TRIP11, and UBE2I) caused a significant decrease in the replication of a DENV replicon. We further showed that calreticulin colocalized with viral dsRNA and with the viral NS3 and NS5 proteins in DENV-infected cells, consistent with a direct role for calreticulin in DENV replication. Human proteins that interacted with DENV had significantly higher average degree and betweenness than expected by chance, which provides additional support for the hypothesis that viruses preferentially target cellular proteins that occupy central position in the human protein interaction network. This study provides a valuable starting point for additional investigations into the roles of human proteins in

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“…2) (73). In this system, putative interacting proteins are expressed as fusions to N-and C-terminal fragments of luciferase.…”

Section: Mapping the Denv-human Protein Interactionmentioning

confidence: 99%

A Physical Interaction Network of Dengue Virus and Human Proteins

Khadka

Vangeloff

Zhang

et al. 2011

Molecular & Cellular Proteomics

153

148

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“…Adopting the nomenclature for RNA splicing, the internal regions of proteins that are "spliced out" have been called inteins and the external, or spliced proteins, are referred to as exteins. The protein slicing mechanism was used to create a reporter gene strategy that would interrogate protein-protein interactions (87). In this study, the approach was to fuse two complementary inteins to two parts of the luciferase such that upon protein-protein interaction, the inteins are excised out of inactive precursor fusion proteins, and the luciferase exteins are ligated into a contiguous luciferase polypeptide with enzymatic activity.…”

Section: Dual Reporter Strategiesmentioning

confidence: 99%

It's not just about anatomy: In vivo bioluminescence imaging as an eyepiece into biology

Contag

Ross

2002

Magnetic Resonance Imaging

254

189

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Among the newly described tools that enable analyses of cellular and molecular events in living animals, in vivo bioluminescence imaging (BLI) offers important opportunities for investigating a wide variety of disease processes. BLI utilizes luciferase as an internal biological light source that can be genetically programmed to noninvasively "report" the presence or activation of specific biological events. Applications of BLI have included the use of luciferase to demonstrate expression of cell-and tissue-specific promoters, label cell populations, guide detection by other imaging modalities, and detect protein-protein interaction. These applications of BLI technology have allowed quantitative measurements of tumor burden and treatment response, immune cell trafficking, and detection of gene transfer. Spatiotemporal information can be rapidly obtained in the context of whole biological systems in vivo, which can accelerate the development of experimental therapeutic strategies. This paper provides a review of the biological applications in which in vivo BLI has been utilized to nondestructively monitor biological processes in intact small animal models, and highlights some of the advancements that will increase the versatility of BLI as a molecular imaging tool.

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“…These split reporters have been used for measuring real time protein-protein interactions efficiently, both in cells and also in living animals. 11,[13][14][15] The inactive protein fragment assisted complementation of Ubiquitin, dihydrofolate reductase, green fluorescent protein, and β-lactamase have been used for studying protein-protein interactions in bacteria and mammalian cells. [16][17][18][19][20][21][22] In our previous studies, we split bioluminescent monomeric proteins firefly luciferase and synthetic renilla luciferase, and the resulting fragments were used for studying protein-protein interactions.…”

mentioning

confidence: 99%

Firefly Luciferase Enzyme Fragment Complementation for Imaging in Cells and Living Animals

2005

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We identified different fragments of the firefly luciferase gene based on the crystal structure of firefly luciferase. These split reporter genes which encode for protein fragments, unlike the fragments currently used for studying protein-protein interactions, can self-complement and provide luciferase enzyme activity in different cell lines in culture and in living mice. The comparison of the fragment complementation associated recovery of firefly luciferase enzyme activity with intact firefly luciferase was estimated for different fragment combinations and ranged from 0.01 to 4% of the full firefly luciferase activity. Using a cooled optical charge-coupled device camera, the analysis of firefly luciferase fragment complementation in transiently transfected subcutaneous 293T cell implants in living mice showed significant detectable enzyme activity upon injecting D-luciferin, especially from the combinations of fragments identified (Nfluc and Cfluc are the N and C fragments of the firefly luciferase gene, respectively): Nfluc (1-475)/Cfluc (245-550), Nfluc (1-475)/Cfluc (265-550), and Nfluc (1-475)/Cfluc (300-550). The Cfluc (265-550) fragment, upon expression with the nuclear localization signal (NLS) peptide of SV40, shows reduced enzyme activity when the cells are cotransfected with the Nfluc (1-475) fragment expressed without NLS. We also proved in this study that the complementing fragments could be efficiently used for screening macromolecule delivery vehicles by delivering to cells stably expressing Nfluc (1-475) and recovering signal. These complementing fragments should be useful for many reporter-based assays including intracellular localization of proteins, studying cellular macromolecule delivery vehicles, studying cell-cell fusions, and also developing intracellular phosphorylation sensors based on fragment complementation.Luciferases are enzymes that emit light in reaction with a specific substrate in the presence of cofactors. A diverse group of organisms use luciferase-mediated bioluminescence to startle predators or to attract prey or mates. The emitted light is used as a detection system for luciferase expression, which acts as a "reporter" for the activity of any regulatory elements that control its expression. Luciferase is particularly useful as a reporter enzyme in living cells and organisms. Firefly luciferase is one among many of the sensitive luciferases and is widely used by researchers to identify different biological events of cells in culture and in living small animals, and it is also used by public health researchers for the detection of food contamination. [1][2][3] The gene encoding firefly luciferase, cloned in 1985 from the North American firefly, Photinus pyralis, is now emerging as the gene of choice for in vivo and in vitro reporting of transcriptional activity in eukaryotic cells. 4 Reporter genes encoding for proteins with optical signatures, either fluorescent or bioluminescent, are a low-cost alternative for realtime analysis of gene expression in small-animal ...

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Split Luciferase as an Optical Probe for Detecting Protein−Protein Interactions in Mammalian Cells Based on Protein Splicing

Cited by 240 publications

References 32 publications

A Physical Interaction Network of Dengue Virus and Human Proteins

A Physical Interaction Network of Dengue Virus and Human Proteins

It's not just about anatomy: In vivo bioluminescence imaging as an eyepiece into biology

Firefly Luciferase Enzyme Fragment Complementation for Imaging in Cells and Living Animals

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