Creation of a caspase-3 sensing system using a combination of split-GFP and split-intein

Sakamoto, Seiji; Terauchi, Mika; Hugo, Anna; Kim, Tanner; Araki, Yasuyuki; Wada, Takehiko

doi:10.1039/c3cc43389g

Cited by 16 publications

(12 citation statements)

References 17 publications

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“…Three groups designed protease sensors that take advantage of split GFP complementation. Each strategy utilizes variations of a cyclic, locked GFP11 fragment that opens upon protease cleavage, allowing for complementation with GFP1–10 and subsequent fluorescence (15, 119, 137). Split GFP also serves as a sensor for protein kinase and phosphatase activity (158) as well as cytoplasmic delivery of cell-penetrating peptides or other cargo (86, 101, 123).…”

Section: Split Fluorescent Proteins and The Detection Of Protein–protmentioning

confidence: 99%

Split Green Fluorescent Proteins: Scope, Limitations, and Outlook

Romei

Boxer²

2019

Annu. Rev. Biophys.

178

161

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Many proteins can be split into fragments that spontaneously reassemble, without covalent linkage, into a functional protein. For split green fluorescent proteins (GFPs), fragment reassembly leads to a fluorescent readout, which has been widely used to investigate protein–protein interactions. We review the scope and limitations of this approach as well as other diverse applications of split GFPs as versatile sensors, molecular glues, optogenetic tools, and platforms for photophysical studies.

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Section: Split Fluorescent Proteins and The Detection Of Protein–protmentioning

confidence: 99%

Split Green Fluorescent Proteins: Scope, Limitations, and Outlook

Romei

Boxer²

2019

Annu. Rev. Biophys.

178

161

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“…The principal bottleneck for cytosolic protein delivery is the dependence on endosomal escape. [21][22][23] GFP-based reporters have been developed to elucidate caspase activity, [24][25][26][27] but to our knowledge, no such development has allowed detection of exogeneous, active or inactive, cytosolic casp-3. Split GFP enables the spontaneous assembly of the GFP beta strands 1-10 (GFP1-10) and the GFP beta strand 11 (GFP11).…”

Section: Introductionmentioning

confidence: 99%

Tracking exogenous intracellular casp‐3 using split GFP

et al. 2020

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Cytosolic protein delivery promises diverse applications from therapeutics, to genetic modification and precision research tools. To achieve effective cellular and subcellular delivery, approaches that allow protein visualization and accurate localization with greater sensitivity are essential. Fluorescently tagging proteins allows detection, tracking and visualization in cellulo. However, undesired consequences from fluorophores or fluorescent protein tags, such as nonspecific interactions and high background or perturbation to native protein's size and structure, are frequently observed, or more troublingly, overlooked. Distinguishing cytosolically released molecules from those that are endosomally entrapped upon cellular uptake is particularly challenging and is often complicated by the inherent pH-sensitive and hydrophobic properties of the fluorophore. Monitoring localization is more complex in delivery of proteins with inherent protein-modifying activities like proteases, transacetylases, kinases, etc. Proteases are among the toughest cargos due to their inherent propensity for self-proteolysis. To implement a reliable, but functionally silent, tagging technology in a protease, we have developed a caspase-3 variant tagged with the 11th strand of GFP that retains both enzymatic activity and structural characteristics of wild-type caspase-3. Only in the presence of cytosolic GFP strands 1-10 will the tagged caspase-3 generate fluorescence to signal a non-endosomal location. This methodology facilitates easy screening of cytosolic vs. endosomallyentrapped proteins due to low probabilities for false positive results, and further, allows tracking of the resultant cargo's translocation. The development of this tagged casp-3 cytosolic reporter lays the foundation to probe caspase therapeutic properties, charge-property relationships governing successful escape, and the precise number of caspases required for apoptotic cell death.

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“…Herein, we optimized and characterized cell-based switch-on fluorescent split GFP sensors conditionally activated by viral proteolytic activity. While comparable sensing strategies were only applied in biochemical or bacterial screening assays [15,17], herein we established mammalian cell-based sensors for different cysteine (TEVp and AVP) and aspartic (HIV-1 PR) viral proteases, and a whole cell biosensing platform of adenovirus infection. The split GFP-based sensors have shown an enormous potential, with SNR up to 97.…”

Section: Discussionmentioning

confidence: 99%

“…A number of split GFP sensors dependent on viral protease activity have been developed [15,16]. Similar strategies were also applied for detection of cellular proteases [17,18]. However, those methodologies require laborious fluorescence analysis, lack throughput, or were not assessed neither in mammalian cells nor, importantly, in response to virus infection.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Structurally Distorted Split GFP Fluorescent Sensors for Cell-Based Detection of Viral Proteolytic Activity

Guerreiro

Fernandes

Coroadinha

2020

Sensors

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Cell-based assays are essential for virus functional characterization in fundamental and applied research. Overcoming the limitations of virus-labelling strategies while allowing functional assessment of critical viral enzymes, virus-induced cell-based biosensors constitute a powerful approach. Herein, we designed and characterized different cell-based switch-on split GFP sensors reporting viral proteolytic activity and virus infection. Crucial to these sensors is the effective—yet reversible—fluorescence off-state, through protein distortion. For that, single (protein embedment or intein-mediated cyclization) or dual (coiled-coils) distortion schemes prevent split GFP self-assembly, until virus-promoted proteolysis of a cleavable sequence. All strategies showed their applicability in detecting viral proteolysis, although with different efficiencies depending on the protease. While for tobacco etch virus protease the best performing sensor was based on coiled-coils (signal-to-noise ratio, SNR, 97), for adenovirus and lentivirus proteases it was based on GFP11 cyclization (SNR 3.5) or GFP11 embedment distortion (SNR 6.0), respectively. When stably expressed, the sensors allowed live cell biosensing of adenovirus infection, with sensor fluorescence activation 24 h post-infection. The structural distortions herein studied are highly valuable in the development of cellular biosensing platforms. Additionally highlighted, selection of the best performing strategy is highly dependent on the unique properties of each viral protease.

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Creation of a caspase-3 sensing system using a combination of split-GFP and split-intein

Cited by 16 publications

References 17 publications

Split Green Fluorescent Proteins: Scope, Limitations, and Outlook

Split Green Fluorescent Proteins: Scope, Limitations, and Outlook

Tracking exogenous intracellular casp‐3 using split GFP

Evaluation of Structurally Distorted Split GFP Fluorescent Sensors for Cell-Based Detection of Viral Proteolytic Activity

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